Pharmaceutical Composition for Improving Health, Cure Abnormalities and Degenerative Disease, Achieve Anti-aging Effect of Therapy and Therapeutic Effect on Mammals and Method Thereof

ABSTRACT

A pharmaceutical composition of the present invention is used for improving health, cure abnormalities and degenerative disease; achieve anti-aging effect of therapy and therapeutic effect on mammals. The pharmaceutical composition includes a pharmaceutical carrier and an isotope selective ingredient including at least one of a chemical element and a chemical compound containing the chemical element whereby isotope distribution in the at least one of the chemical element and the chemical compound containing the chemical element is different from natural distribution of at least one of isotopes wherein the part of selected isotope of the chemical element ranges from 0 to 100%. A method of the present invention uses the inventive pharmaceutical composition to improve health, cure abnormalities and degenerative disease and achieve therapeutic effect on mammals.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/613,109, filed on Jun. 2, 2017, which is a continuation-in-part ofU.S. patent application Ser. No. 14/833,114, filed on Aug. 23, 2015,which claims the benefit of U.S. provisional application Ser. No.62/123,900, filed on Dec. 1, 2014, and entitled “SYSTEM, APPARATUS.METHODS AND COMPOSITIONS FOR THE TREATMENT OF GROUP OF DISEASESINVOLVING ABNORMAL CELL GROWTH AND OTHER HEALTH ABNORMALITIES OFMAMMALS,” all of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition forimproving health, cure abnormalities and degenerative disease, includingsuppression of the development of cancer, for example, suppressing thegrowth of tumors (solid and non-solid) and suppressing, partially orcompletely, the spread of cancer, i.e. metastasis; achieve anti-agingeffect of therapy and therapeutic effect on mammals and method ofadministering the same.

BACKGROUND OF THE INVENTION

Cancer is one of the degenerative diseases. Cancer is characterizedprimarily by an increase in the number of abnormal cells derived from agiven normal tissue, invasion of adjacent tissues by these abnormalcells, or lymphatic or blood-borne spread of malignant cells to regionallymph nodes and to distant sites (metastasis). Clinical data andmolecular biologic studies indicate that cancer is a multistep processthat begins with minor preneoplastic changes, which may under certainconditions progress to neoplasia. The neoplastic lesion may evolveclonally and develop an increasing capacity for invasion, growth,metastasis, and heterogeneity, especially under conditions in which theneoplastic cells escape the host's immune surveillance. There is anenormous variety of cancers which are described in detail in the medicalliterature. Examples include cancer of the lung, colon, rectum,prostate, breast, brain, and intestine.

At present, cancer is the second leading cause of death globally, aftercardiovascular diseases. Statistics show that the incidence ofoncological diseases is growing every year, and today this problem isone of the greatest challenges for medicine and the pharmaceuticalindustry.

The art is replete with various compositions and methods for treatingcancer. One of such compositions is disclosed in US Patent ApplicationPublication Number US 2014/0219961A1 to Jung et al. entitled“Pharmaceutical composition for treating cancer, comprising interferonalpha conjugate”. The US Patent Application Publication NumberUS20140219961A1 to Jung et al., teaches a method for preventing ortreating a cancer includes administering an anti-cancer pharmaceuticalcomposition including an interferon alpha or a polymer conjugatethereof. The pharmaceutical composition can be co-administered withanti-cancer agents. The interferon alpha conjugate shows a longer invivo half-life and a more excellent anti-cancer activity than theconventional interferon alpha, and in particular, its co-administrationwith an anti-cancer agent such as gemcitabine has synergistic inhibitoryeffects on cancer cell growth and proliferation so as to exhibit aremarkably excellent anti-cancer activity.

Another prior art reference, namely U.S. Pat. No. 8,846,630 to Won etal. entitled “Pharmaceutical composition for treating cancer”. Itdiscloses a pharmaceutical composition for treating cancer, comprisingat least one selected from deoxyribonucleic acids (DNA) for encodingsmall interfering RNA (siRNA) which complementarily binds to the basesequence of the transcript (mRNA transcript) of the FLJ25416 gene,represented by sequence number 3, sequence number 5, and sequence number7 to inhibit the intracellular expression of the FLJ25416 gene,antisense RNA which inhibits expression of the FLJ25416 gene, and shorthairpin RNA (shRNA) which inhibits expression of the FLJ25416 gene. Asthe siRNA, which is complementary to the base sequence of the transcript(mRNA transcript) of the FLJ25416 gene, the antisense RNA, and theshRNA, according to the present invention, inhibit expression of theFLJ25416 gene which is known to be expressed in cancer cells, and thuskill cancer cells, the composition of the present invention can be usedas a novel anti-cancer agent.

Today oncological diseases are mainly treated by chemotherapy that usesa number of chemotherapeutic drugs that are divided into several majorgroups (classes), such as alkylating agents, antimetabolites, antitumorantibiotics, alkaloids. Other biological active substances of plantorigin, as well as enzyme and hormone preparations are also used incancer treatment.

But these drugs are usually not very effective in treating the malignantascites which is associated with the terminal stage of the disease andis very difficult to treat. Malignant ascites is a pathologicalaccumulation of fluid in the peritoneal or pleural cavity that developsas a result of a malignant process in the peritoneum or lungs. Ascitesleads to various functional disorders in patients with advanced stagecancer and is a complex clinical problem. Malignant ascites can becaused by a variety of primary tumors, such as, for example, breastcancer, ovarian cancer or gastrointestinal carcinoma. Despite the factthat in most patients ascites is found already at the firstmanifestation of tumor disease, its appearance indicates the progressionof cancer.

In oncology, cancers that cause ascites pose a serious problem, as inmost cases they result in the death of patients. The death of suchpatients occurs even in the absence of distant metastases and tumorprogression. To treat such types of cancer, conventional types of cancertreatment are used, which to some extent improve the condition andprolong the life of a patient. The available options of therapy forascites carcinomas include puncture, local chemotherapy or treatmentwith diuretics. All these options have significant drawbacks. Thus,puncture only results in short-term relief and should be repeated onaverage every 9.5 days (Mackey et al., J. Pain Symptom Manage, 19: 193,2000). Chemotherapy can also be successful only in patients who have notyet developed tumor cells resistant to chemotherapy, which,unfortunately, occurs quite often. In particular, after the puncture isperformed, intraperitoneal chemotherapy is used to remove exudates byadministration of cytostatic agents such as adriamycin, thiotepa,cyclophosphamide, doxorubicin, paclitaxel, docetaxel, topotecan, etc.,as well as their combinations (Brenner D E, Intraperitonealchemotherapy: a review, J Clin Oncol, 1986, 4 (7): 1135-1147). At thesame time, the peritoneum actively absorbs chemotherapeutic drugs, andthey enter the bloodstream in significant concentrations. Thusmetastases in the abdominal cavity directly contact the cytostaticagent, the tumor cells circulating in the blood are destroyed and themedicinal effect on all cancer sites in the body is produced. However, adisadvantage of such treatment is its relatively low efficiency with ashort-term palliative effect.

In certain cases, platinum-based drugs, such as carboplatin andcisplatin Meshcheryakova N.G. Platinum cytostatics in the treatment oftumor pleurisy. MD Dissertation, M., 1993), are used to treat ascitescaused by the tumor process. These chemical compounds are complexinorganic compounds of transition metals (platinum), in contrast to theprevailing majority of antitumor drugs which according to their chemicalstructure are organic substances as a rule. The complex compounds ofplatinum include a central ion, or a complexing agent, which is divalentplatinum surrounded by monodentate ligands, two of which are chlorineatoms and two are molecules of ammonia, in the cis-position, i.e. theseligands are located in square-built complexes on one side of thecomplexing agent. The geometric configuration of platinum complexesplays an important role in their biological activity. According to itsphysical and chemical properties, cisplatin is a neutral complex ofplatinum, which facilitates its transport through cell membranes. Thechemical mobility of substitutable groups is quite high, and thiscreates good conditions for bonds of important biochemical substrates inthe body with the nucleophilic centers. Among disadvantages of themethods using compositions comprising platinum compounds are increasednephrotoxicity and hepatotoxicity. In addition, methods that involve theuse of cisplatin cause oppression of normal hematopoiesis, neuropathies,electrolyte disorders or reversible increase in the activity ofaminotransferases. In addition, these methods do not completely stop thedevelopment of ascites, but only slow its growth to a certain extent.

It has been demonstrated by a large number of studies that the isotopiccomposition oftissues and organs can serve as a diagnostic marker. Inparticular, the study of the ratios of Cu and Zn isotopes in bloodshowed their promising interrelationships with age, sex and pathologies.For example, an estimate of the ratio of Cu isotopes in blood serum is anew approach to the diagnosis and prognosis of the development ofcirrhosis (M. Costas-Rodriguez, Y. Anoshkina, S. Lauwens, H. VanVlierberghe, J. Isotopic analysis of Cu in blood serum bymulti-collector ICP-mass spectrometry: a new approach for the diagnosisand prognosis of liver cirrhosis, Metallomics 2015, 7. 491-498), and theisotopic composition of Zn in breast tissues enables diagnosis of cancer(F. Larner, L. N. Woodley, S. Shousha, A. Moyes, E. Humphreys-Williams,S. Strekopytov, A. N. Halliday, M. Rehkamper, R. C. Coombes, Zincisotopic compositions of breast cancer tissue, Metallomics 2015, 7.107-112).

WO2001082871 discloses a method for therapy and diagnosis of coloncancer using a composition based on zinc isotopes having a shorthalf-life, ⁶²Zn in particular, selected from the group consisting ofzinc acetate, zinc chloride, and zinc sulfate, to induce apoptosis inthe tumor cells of the large intestine. As it is demonstrated therein,the composition containing ⁶²Zn at a concentration of 60 to 80 μM, inthe presence of phosphate binder, induces apoptosis in colon cancercells. However, the data presented in the said publication relate onlyto the in vitro experiments on HT-29 cell lines (ATCC cell line numberHTB 38) and T-84 (ATCC cell line number CCL 248) derived from a humancolon tumor.

Another known method for inhibiting a malignant process is theemployment of compositions comprising nanoparticles ofporphyrinfulfullerenes (NP) containing such isotopes as ²⁵Mg and ⁶⁷Zn(²⁵Mg—NP and ⁶⁷Zn—NP) (Orlova, M. A.; Osipova, E. Yu.; Rumyantsev, S.A.; Ashurko, S. P. Effect of the ⁶⁷Zn isotope on leukemic cells andnormal lymphocytes.—Russian Chemical Bulletin (2012), 61(2), 405-408).When using the described method and compositions, significantdifferences in the cytotoxic effect of magnetic and nonmagnetic zincisotopes on tumor cells were observed, as well as the lack of effect ofthe complex of magnetic magnesium isotope and primary nanoparticles onsuch cells. ⁶⁷Zn—NP showed potent cytotoxic activity against cells ofacute B-lymphoblastic leukemia with LD50 almost three times lower thanin healthy donors and four times lower than when using Zn—NP. However,as in the previous case, the experiments were performed in vitro onhuman leukemia cells, towards which a cytotoxic effect was observed. Theefficacy of the described composition and method has not been evaluatedin animal models or in a clinical trial.

However, the compositions and methods described in the prior artdocuments mentioned above, which involve the use of light isotopes ⁶²Znor ⁶⁷Zn, differ from the claimed ones by the zinc isotope used, diseasesthat can be treated with it, and additional active ingredients thatprovide an inhibitory effect on tumors that cause ascites.

In addition to cancer, other chronic and degenerative diseases havebecome primary cause of mortality. Neoplasms, ischemic heart disease,and cerebrovascular disease cause three-quarters of all deaths at 35-69years of age and two-thirds at older ages. Huge resources are committedto the reduction of mortality from leading killers. There is no doubtthat eventually dramatic results will follow in this direction.

The problem is that most of the degenerative pathologies such as heartdisease, cancer and Alzheimer disease are age related, and those atfirst glance are not considered being results of a specific tissue“local” aging. Therefore as best formulated by George Johnson/NY TimesJan. 4, 2014/ “ . . . barring an elixir for immortality, a body willcome to a point where it has outwitted every peril life has thrown atit. And for each added year, more mutations will have accumulated. Ifthe heart holds out, then waiting at the end will be cancer.” As long asaging is not addressed properly, we can only hope for partiallysuccessful treatments that only pushing problem a little bit further intime, still failing to avoid final negative outcome.

When dealing with infectious diseases, the cause is clear. In case ofdegenerative diseases, there is much less clarity. Over the last hundredyears, the focus of research was moving along the line“organ-tissue-cell-molecule/protein” providing precious data needed tocrate new medicine. Yet in most cases, we have to evaluate success interms of statistical outcome in treating not the cause but syndromes ofdisease. To change the current trend, we need a solution that provides aresistance to bacterial and virus attacks and should be able to repairdamage from critical mutations on molecular level. Regenerate/renew“defective” tissue damaged by chronic degenerative disease. Preventtransformation of adult stem cells into cancer stem cells. Transformcancer cells into benign tissue cells or harmless stem-like cells.Prevent health deterioration in time on structural level and byincreasing efficiency of immune system.

The current paradigm in cancer research is in the following statement:“cancer is a disease of cells, and the phenotypes of cancer cells can beunderstood by examining the genes and proteins within them”.Correspondently one of the main problems scientists trying to address ishow many intracellular regulatory circuits need to be perturbed in orderto transform a normal human cell into a cancer cell?

Therefore, not only cancer research but any other pathologyinvestigation will stop there.

The question is whether it's good enough or not. It is perfectly fine iflife exists due to a lucky coincident of random events. But what if lifeas we know it is a specific form of a universal algorithm expressed andevolving at given physical conditions here and now? We often speak interms of mutations, genetic defects, DNA code. Well, behind every codemust be an algorithm and behind any algorithm must be mathematics. If itis true, then the knowledge of it could be a precondition for successfulcontrol/correction of any life form.

There are 200 different types of cells in human body. Let us imaginethat in each cell we select the same chromosome and purposely damage it.It may lead to the development of the disease in different cells,tissues, organs having different symptoms and even named differently,all due to one single type of damage given said damage is “essential”.So, what defines the severity of the damage? There is a big variety ofknown pathogens including bacteria, viruses, chemicals, radiation etc.Irreversible damage may happen without a pathogen as well, such asmutation in the process of mitosis. At the same time, most of themistakes during cell division are immediately repaired.

Infection diseases are sometimes defeated by immune system withoutirreversible changes in the organism. What may go wrong and where? Whichchange has a potential to become a fatale one? What is the first,elementary step of pathological change caused by any pathogen withoutexception? Life scientists would start answering with the reference toprocesses in the cells. However, it is the wrong level.

The fact is that we can completely ignore any pathogens as long as not asingle chemical bond in the organism is damaged. It means that theinteraction between pathogen and organism did not take place. When itdoes, a lot depends on the type of chemical bond that was destroyed andwhat have happened in the result. A lot of heavy smokers and alcoholconsumers do not get cancer, but some of those with healthy life styleare less lucky. Why? We need to understand that if damages caused bypathogens were limited to a certain level of chemical bond, then theywould be repaired by internal mechanisms the same way as reparation ofmistakes during mitosis happens.

The shocking truth is that damage sometime goes deeper than chemicalbonds level—to the nucleus. Once it happens, there is no way back, andchange becomes irreversible contributing to the unavoidable finalewhether it is about illness or aging. Any efforts to change thesituation on the level above the atom will be fruitless and in the bestcase capable to address just symptoms of diseases.

It is not enough to think in terms of homeostasis. It is essential tounderstand that homeostasis starts from elementary particles and goes upto the levels higher than individual mind or intellect and not limitedby the currently agreed borders of life science. Inability of human (andall animals in general) as a stable system to control changes on atomiclevel allows to suggest that animals including humans were originated inthe different and in a certain aspect artificial or selectiveenvironment. The one where damage of chemical bonds would never lead tothe changes on atomic or sub-atomic levels and as a result the originalhierarchical structure would never be jeopardized.

While not wishing to be bound by theory, the present inventors believethat certain elements, including each of potassium, magnesium, zinc,rubidium, silicon, calcium, copper, iron, chromium, nickel, molybdenum,selenium, bromine, and chlorine, play important roles in autocatalyticreactions in the body of an animal, such as a human or other mammal. Theproducts of such autocatalytic reactions, such as proteins, playimportant chemical and structural roles in the body, including immunefunction. Fully functional products of such reactions require aspecific, “correct” chirality at various chiral centers within theproduct. The inventors further understand that heavy isotopes accumulatein the body beginning at birth such that, over time, the relativeabundance of each element's isotopes drifts further and further from thenaturally occurring relative abundance, becoming increasinglyover-weighted with respect to heavy isotopes. Heavy isotopes can affectautocatalytic reactions by reducing the proportion of products that havethe “correct” chirality. See, e.g., Tsuneomi Kawasaki et al., AsymmetricAutocatalysis Triggered by Carbon Isotope (¹³C/¹²C) Chirality, Science324: 492-95 (2009). This causes a reduction in the proportion ofproducts of autocatalytic reactions that are fully functional. In sum,the cumulative divergence of the body's isotope relative abundances fromthe natural relative abundance causes a decrease in the functionality ofvarious proteins and other molecules in the body, leading to a declinein health with age.

The present inventors believe such a decline can be countered byrestoring the body's original isotope relative abundances, or by movingthe isotope relative abundances in that direction. This can be achievedin accordance with the present invention by administering one or more ofthe above-listed elements enriched (relative to its natural abundance)with a corresponding “light isotope,” specifically, enriched with ³⁹K,²⁴Mg, ⁶⁴Zn, ⁸⁵Rb, ²⁸Si, ⁴⁰Ca, ⁶³Cu, ⁵⁴Fe, ⁵²Cr, ⁵⁸Ni, ⁹²MO, ⁷⁴Se, ⁷⁹Br,³⁵Cl, respectively, to a patient (human or non-human animal), which canalter the chirality of the autocatalytic products present in thepatient, resulting in an improvement in the patient's health. Further,the quantity of light isotope that is effective may be proportional tothe quantity of the corresponding element that is present in the body.Where the body contains a relatively large quantity of the element, acorrespondingly relatively large amount of the element's light isotopewill be required to provide an effective dosage amount.

On the other hand, where the body contains a relatively small quantityof the element, a correspondingly relatively small amount of theelement's light isotope will be required to provide an effective dosageamount.

The incidence of cancer continues to climb as the general populationages, as new cancers develop, and as susceptible populations (e.g.,people infected with AIDS or excessively exposed to sunlight) grow. Atremendous demand therefore exists for new methods and compositions thatcan be used to treat patients with cancer.

SUMMARY OF THE INVENTION

A pharmaceutical composition of the present invention is used forimproving health, cure abnormalities and degenerative disease; achieveanti-aging effect of therapy and therapeutic effect on mammals. Thepharmaceutical composition includes a pharmaceutical carrier and anisotope selective ingredient including at least one of a chemicalelement and a chemical compound containing the chemical element wherebyisotope distribution in the at least one of the chemical element and thechemical compound containing the chemical element is different fromnatural distribution of at least one of isotopes wherein the part ofselected isotope of the chemical element ranges from 0 to 100%. Theselected isotopes include at least one of K-39; Mg-24; Zn-64; Rb-85;Si-28 and combination thereof. The selected isotopes also include atleast one of Ca-40; Cu-63; Fe-54; Cr-52; Ni-58; Mo-92; Se-74; Br-79;Cl-35 and combination thereof. The pharmaceutical of the carrierpharmaceutical composition is used in the form of a solution, a gel, acream, a spray, an aerosol, a patch, nanoparticles, inorganic molecules,organic molecules, a plant, a fruit and a vegetable.

One specific technical problem to be solved by the present invention isto provide a composition comprising an active ingredient and a methodfor suppression of the development of cancer, including suppressing thegrowth of tumors (solid and non-solid) and suppressing, partially orcompletely, the spread of cancer, i.e. metastasis, wherein the methodentails administering the composition, which possesses anticanceractivity and also does not produce side effects as toxic as thoseassociated with the use of known cytostatic agents. The composition ofthe invention generally is a pharmaceutical composition that contains asan active ingredient one or more light-isotope enriched elementsselected from ⁶⁴Zn-enriched zinc, ³⁹K-enriched potassium, SRb-enrichedrubidium, ²⁴Mg-enriched magnesium, ⁵⁴Fe-enriched iron, ⁷⁴Se-enrichedselenium, ²⁸Si-enriched silicon, ⁴⁰Ca-enriched calcium, and⁶³Cu-enriched copper, either in elemental form or, preferably, in theform of a chelate, salt, complex, or other pharmaceutically acceptablecompound. The composition preferably contains at least one excipient.

The pharmaceutical composition includes combination of at least two ofthe isotopes wherein one of the isotopes is lighter in weight than theother of the isotopes to achieve therapeutic effect. The light isotopesof the pharmaceutical composition are K-39; Mg-24; Zn-64; Rb-85; Si-28;Ca-40; Cu-63; Fe-54; Cr-52; Ni-58; Mo-92; Se-74; Br-79; Cl-35. Thechemical compounds of the pharmaceutical composition include theisotopes such as at least one of oxides, sulfates, citrates, gluconate,and a chelate containing a ligand bonded to a central metal atom atleast two points. The chemical elements and chemical compounds are foodsupplements. (Throughout this application, isotopes are designatedinterchangeably two different ways: for example, ³⁵Cl and Cl-35 refer tothe same chlorine isotope.) In another embodiment, the compositioncomprises an effective therapeutic amount of at least one light isotopeselected from the group consisting of K-39, Mg-24, Zn-64, Rb-85, Si-28,Ca-40, Cu-63, Fe-54, Cr-52, Ni-58, Mo-92, Se-74, Br-79, and Cl-35 eitherin elemental form or in the form of a pharmaceutically acceptable salt,compound, chelate, or complex, wherein the composition is enriched forthe at least one light isotope relative to the natural abundance of theisotope. In preferred embodiments, the composition is suitable forvarious routes of administration, such as topical or oral administrationor administration by injection. In certain embodiments, the compositionfurther comprises at least one additional ingredient suitable to theform of the composition, including carriers and excipients such asdiluents, solvents (such as water), binders, lubricants, coloringagents, and preservatives, which are conventional and known to theperson of ordinary skill in the art. The composition preferably isformulated for a specific route of administration such as, but notlimited to, injection (e.g. intravenous, intraperitoneal, orsubcutaenous injection), topical administration and oral administration,and other parenteral routes not mentioned above (e.g. via suppository).Other conventional routes of administration may also be used asappropriate to the condition being treated. Specific exemplary forms ofthe composition include a topical solution, spray, lotion, salve,ointment, gel, cream, soap, shampoo, patch, powder and foam, and an oraltablet, capsule, syrup, suspension, lozenge, gum, spray, and solution,and a solution or other composition suitable for intravenous,intraperitoneal, subcutaneous, or other route of administration byinjection. In one embodiment, the light isotope is packaged inliposomes, which in turn are dissolved or suspended in an appropriateliquid and packaged in capsules that are administered orally. In otherembodiments, oral compositions of the invention may be formulated forimmediate, delayed, or sustained release and may also be formulated forenteric release. Topical compositions of the invention may include atleast one absorption-enhancing agent such as DMSO.

Compositions in the form of a solution (for any appropriate route ofadministration, including by injection) may be prepared in which thesolvent is water; in a preferred embodiment, the water is enriched with¹⁶O and/or depleted of ²H, both with respect to the isotopes' naturalabundance. For example, the hydrogen of the deuterium-depleted water maybe at least 99.99% ¹H on a mole fraction basis. Further, the oxygen ofthe deuterium-depleted water may be at least 99.9% ¹⁶O on a molefraction basis. In an embodiment, the composition also containscompounds that promote better penetration of the agent into body cells.In a preferred embodiment, the at least one light isotope that thecomposition is enriched for is one or more of ⁶⁴Zn, ²⁴Mg, ³⁹K, ⁵⁴Fe,⁸⁵Rb, and ²⁸Si. In preferred embodiments, in a solution as describedabove, ⁶⁴Zn_(e) is present at a concentration of from 10 μg/ml to 10mg/ml, most preferably from 100 μg/ml to 2 mg/ml; in these embodiments,⁶⁴Zn_(e) is present in the form of a chelate of an amino acid,preferably a chelate of aspartate or asparaginate.

When the enriched-for light isotope is ⁶⁴Zn, the ⁶⁴Zn preferablyconstitutes at least about 80% ⁶⁴Zn of the zinc on a mole fraction basis(that is, 80% of the zinc atoms are ⁶⁴Zn atoms), more preferably atleast about 90% ⁶⁴Zn, such as at least 90%, at least 95%, or at least99% ⁶⁴Zn, or between about 90% and about 99.9% ⁶⁴Zn (all on a molefraction basis). When the enriched-for light isotope is ⁶⁴Zn, the⁶⁴Zn-enriched zinc (“⁶⁴Zn_(e)”) preferably is present as a chelate of anamino acid, such as of asparaginate, aspartate or glutamate.⁶⁴Zn-enriched zinc may also be provided, for example, in the form of asalt or chelate with sulfate, citrate, or ethylene diamine disuccinicacid (referred to herein both as “EDDA” and as “EDDS”).

In alternative embodiments, any of the above compositions can compriseas a therapeutic agent at least one light isotope selected from anysubgroup selected from the group consisting of K-39, Mg-24, Zn-64,Rb-85, Si-28, Ca-40, Cu-63, Fe-54, Cr-52, Ni-58, Mo-92, Se-74, Br-79,and Cl-35, each independently either in elemental form or in the form ofa pharmaceutically acceptable salt, compound, chelate, or complex,wherein the composition is enriched for the at least one light isotoperelative to the natural abundance of the isotope.

In various embodiments, the light isotope in the composition of theinvention is present in elemental form or in the form of one or more ofan oxide, sulfate, citrate, gluconate, chelate, or other compound, or inany other pharmaceutically acceptable form. The at least one lightisotope may be present in the composition in the form of a salt orchelate with a pharmaceutically acceptable inorganic or organic acid.Exemplary salts and chelates of the light isotope include the sulfate,glutamate, asparaginate, aspartate, citrate, and ethylene diaminedisuccinate of the light isotope. Preferred chelates and salts of thelight isotope are formed in combination with an amino acid generally,preferably an amino acid that occurs naturally, such as one of thetwenty amino acids that occur in the vast majority of proteins.

In an embodiment, the composition of the invention further comprises anactive ingredient in addition to the light isotope active ingredient. Inan embodiment, the composition of the invention comprises an agent thatenhances the stability of the composition.

The light isotope may constitute between about 0.1% and about 99% of thecomposition by weight. When the light isotope is present in the form ofa salt, the anionic portion of the salt acts as a carrier. When water ispart of the said composition, it may function as a carrier and diluent.

The composition of the invention can be used in medicine to treat humansand non-human animals, including veterinary mammals. The compositionsdescribed above can be used in the methods detailed below.

A method of using the pharmaceutical composition to improve health, cureabnormalities and degenerative disease and achieve therapeutic effect onmammals is provided. The method begins with preparing the pharmaceuticalcarrier and the isotope selective ingredient including at least one ofthe chemical element and the chemical compound containing the chemicalelement whereby isotope distribution in the at least one of the chemicalelement and the chemical compound containing the chemical element isdifferent from natural distribution of at least one of isotopes whereinthe part of selected isotope of the chemical element ranges from 0 to100%.

The next step of the method includes administering the first of theisotopes at least prior to and after surgical removal of a solid tumorto prevent possible metastases and occurrence of secondary effects andto prevent metastasizing followed by administering a second of theisotopes to transform a cancer cell phenotype into a normal cell. Thefirst of the isotopes administered prior to and after surgical removalof the solid tumor to prevent possible metastases include at least oneof K-39; Mg-24; Zn-64; Rb-85; Si-28; Ca-40; Cu-63; Fe-54; Cr-52; Ni-58;Mo-92; Se-74; Br-79; Cl-35 and combination thereof. The second of theisotopes used to transform the cancer cell phenotype into the normalcell includes at least one of K-39, Mg-24, Zn-64, Rb-85, Si-28 andcombination thereof. The step of administering the pharmaceuticalcarrier can be carried out orally, intravenously and locally withoutlimiting the scope of the present invention. The isotope selectiveingredient may be administered prior to and after surgical removal of asolid tumor to prevent further spreading of cancer cells andmetastasizing of a primary tumor. The chemotherapeutic agent includes atleast one of Actinomycin, All-trans retinoic acid, Azacitidine,Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine,Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin,Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone,Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib,Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan,Valrubicin, Vinblastine, Vincristine, Vindesine, and Vinorelbine. Themethod of the present invention allows administering the isotopeselective ingredient to cause fast and significant reduction in degreeof malignancy and to induce changes of cells phenotype form malignantphenotype to a benign or normal phenotype.

The isotope selective ingredient administered at least prior to, afterand simultaneously with chemotherapeutic agent are used to amplifytherapeutic effect on cancer tissue and to protect healthy tissue fromchemotherapy side effects and from immunotherapy side effect. The methodof the present invention allows administering the isotope selectiveingredient to cause fast and significant reduction in degree ofmalignancy and to induce changes of cells phenotype form malignantphenotype to a benign or normal phenotype. The person skilled in the artcan provide shifting the balance in favor of youth and health with rightcombination of light isotopes in optimal doses. In general, isotopecompositions and doses are always case specific. For the purpose ofdiseases and aging prevention, daily intake doses are sufficient. Fortherapeutic treatment much higher doses like triple daily intake aremore suitable.

In another embodiment, the invention provides a method for suppressingthe development of malignant ascites in a human or animal patient (e.g.a veterinary animal) and also provides a method for the treatment ofcancers causing ascites and/or the suppression of cancer metastasis bothin humans and in animals. Alternatively viewed, the invention provides amethod of treating cancer, such as solid and non-solid tumors, and ofpreventing, suppressing, or reducing the extent of metastasis. Themethod comprises the step of administering the composition of theinvention, described above, to a patient in need of such treatment by aroute suitable to the form of the composition and the form of cancerbeing treated, e.g. orally, topically, or by injection (such asintravenous injection, intraperitoneal injection, or injection into thetumor itself). In a preferred aspect, the composition is a liquidcomposition, e.g. an aqueous solution as described above, and the methodcomprises administering the liquid composition via intraperitonealinjection, intravenous injection or injection into the tumor. It ispreferred that the administration of the liquid composition beintraperitoneal. The dose of the light isotope-enriched compound mayvary depending on the severity of the disease, the condition of thepatient and other factors that will be considered by a practitionerskilled in the art when determining the dosage and administration routefor the particular patient. In preferred embodiments of the method, thecomposition comprises an effective amount of one or more of⁶⁴Zn-enriched zinc, ²⁴Mg-enriched magnesium, ⁸⁵Rb-enriched rubidium,³⁹K-enriched potassium, and ⁴Fe-enriched iron. In preferred embodiments,the cancer treated is breast cancer or leukemia.

Both oral and intravenous administration of pharmaceutical compositionsare considered as efficient. Ideally, the supply/intake of any heavyisotopes (especially K, Zn and Mg) should be excluded for as long aspossible, but at least several hours prior and after administration ofmedicine. Food with natural distribution of isotopes to be avoided. Toget better and staying healthy requires constant isotope-selectivetreatment or diet. Taking into consideration current situation withprice and availability of light isotopes—for as long as practical. Invivo experiments results demonstrate that isotope-selective treatmentallows for transformation of pathology affected cells into ones withnormal or close to normal phenotype in the matter of days.

An advantage of the present invention is to provide a pharmaceuticalcomposition and a method of using the pharmaceutical composition toimprove health, cure abnormalities and degenerative disease and achievetherapeutic effect on mammals.

Another advantage of the present invention is to provide thepharmaceutical composition and the method of using the pharmaceuticalcomposition to amplify therapeutic effect on cancer tissue and toprotect healthy tissue from chemotherapy side effects and fromimmunotherapy side effect.

Still another advantage of the present invention is to provide thepharmaceutical composition and the method of using the pharmaceuticalcomposition to allow administering the isotope selective ingredient tocause fast and significant reduction in degree of malignancy and toinduce changes of cells phenotype form malignant phenotype to a benignor normal phenotype.

Still another advantage of the present invention is to provide thepharmaceutical composition and the method of using the pharmaceuticalcomposition to allow administering the first of the isotopes at leastprior to and after surgical removal of a solid tumor to prevent possiblemetastases and occurrence of secondary effects and to preventmetastasizing followed by administering a second of the isotopes totransform a cancer cell phenotype into a normal cell. The first of theisotopes administered prior to and after surgical removal of the solidtumor to prevent possible metastases.

These and other objects of the present invention will become readilyapparent upon further review of the following specification anddrawings.

Other objects, features, and advantages of the present invention willbecome apparent upon consideration of the following detailed descriptionof a preferred embodiment thereof, when taken in conjunction with theappended drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1A is a schematic view of a pharmaceutical composition of thepresent invention used for improving health, cure abnormalities anddegenerative disease, achieve anti-aging effect of therapy andtherapeutic effect on a mammal;

FIG. 1B illustrates a Table of Concentration Characteristics ofComponents;

FIG. 2 illustrates results of Immunocytochemical Analysis of AdhesionProteins and Cytoskeleton of N-cadherin and ICAM in Namalwa Cells afterthe Action of Components Containing ³⁹K, ⁶⁴Zn and ²⁴Mg;

FIGS. 3A-3D illustrate an expression of N-cadherin in Namalwa cells;

FIGS. 4A-4D illustrate an expression of CD44 marker in Namalwa cells;FIG. 4A: control cells (no influence of the components), FIG. 4B: cellsafter the action of component containing ³⁹K at a dose of 2 mg/ml, FIG.4C: cells after the action of component containing ⁶⁴Zn at a dose of 10mcg/ml, FIG. 4D: cells after the action of component containing ²⁴Mg ata dose of 2 mg/ml (lens×100);

FIGS. 5A-5F illustrate an expression of ICAM in Namalwa cells; FIG. 5A:control cells (no influence of the components), FIG. 5B: cells after theaction of component containing ³⁹K at a dose of 2 mg/ml, FIG. 5C andFIG. 5D: cells after the action of component containing ⁶⁴Zn at a doseof 10 mcg/ml, FIG. 5E and FIG. 5F: cells after the action of componentcontaining ²⁴Mg at a dose of 2 mg/ml (lens×100);

FIG. 6 illustrates results of Immunocytochemical Analysis of AdhesionProteins and Cytoskeleton of CD44 and IgM in Namalwa Cells after theAction of Components Containing 39K, ⁶⁴Zn and ²⁴Mg;

FIG. 7 illustrates results of Immunocytochemical Analysis of AdhesionProteins and Cytoskeleton of N-cadherin, ICAM and CD44 in HL-60 Cellsafter the Action of Components Containing ³⁹K, ⁶⁴Zn and ²⁴Mg;

FIGS. 8A-8H illustrate an expression of CD44 in HL-60 cells; FIG. 8A andFIG. 8B: control cells (no influence of the components), FIG. 8C andFIG. 8D: cells after the action of component containing ³⁹K at a dose of2 mg/ml, FIG. 8E and FIG. 8F: cells after the action of componentcontaining ⁶⁴Zn at a dose of 10 mcg/ml, FIG. 8G and FIG. 8H: cells afterthe action of component containing 2⁴Mg at a dose of 2 mg/ml (lens×100);

FIGS. 9A-9H illustrate an expression of ICAM in HL-60 cells; FIG. 9A andFIG. 9B: control cells (no influence of the components), FIG. 9C andFIG. 9D: cells after the action of component containing ³⁹K at a dose of2 mg/ml, FIG. 9E and FIG. 9F: cells after the action of componentcontaining ⁶⁴Zn at a dose of 10 mcg/ml, FIG. 9G and FIG. 9H: cells afterthe action of component containing ²⁴Mg at a dose of 2 mg/ml (lens×100);

FIGS. 10A-10L illustrate Morphological and growth characteristics ofcells of HL-60 cell line after their treatment with componentscontaining ³⁹K, ⁶⁴Zn and ²⁴Mg;

FIG. 11A and FIG. 11B illustrates views of Spectophotometer (FIG. 11A)and samples (FIG. 11B) prepared for the analysis after they were treatedwith components with the natural isotope distribution;

FIG. 12 illustrates renal cell carcinoma (PA) and tumor fragment withextensive polymorphism, lymphoid cells and blood capillaries;

FIG. 13 illustrates an animal weight on the 29th day after inoculationwith the renal cell carcinoma (PA);

FIG. 14 illustrates a tumor growth chart built on the results ofobservations of an animal with the transplanted tumor for 29 days, whichchart clearly describes the kinetics of the tumor growth (FIG. 3, 4);

FIG. 15 illustrates kinetics of PA tumor growth in the laboratoryanimal;

FIG. 16 illustrates an initial cells of the tumor strain of renal cellcarcinoma (PA) in a rat;

FIG. 17 illustrates two cell types after treating them with ⁶⁴Zn, ²⁴Mgand ³⁹K. Staining method—Trypan blue;

FIG. 18 illustrates a cell membrane is not damaged, cells of type B;

FIG. 19 illustrates cells of type A; FIG. 19A and FIG. 19B illustratecells of type A;

FIG. 20 illustrates PA tumor cells after treating them with the elementswith natural isotope distribution. Dark (dead) and white (living) cells;

FIG. 21 illustrate initial cell suspension in the plates in twoconcentration models; FIG. 21A and FIG. 21B illustrate initial cellsuspension in the plates in two concentration models;

FIG. 22 illustrates an effect of ³⁹K, ⁶⁴Zn and ²⁴Mg on cells of strainof renal cell carcinoma in the in vitro experiment, experiment with 30000 cells;

FIG. 23 illustrates an effect of ³⁹K, ⁶⁴Zn and ²⁴Mg on cells of strainof renal cell carcinoma in the in vitro experiment Experiment with 300000 cells;

FIG. 24 illustrates an interrelation between quantitativecharacteristics of ⁶⁴Zn, ²⁴Mg and ³⁹K and a number of cells of type A;

FIG. 25 illustrates an effect of K, Zn and Mg elements with naturalisotope distribution on the number of viable cells in the in vitroexperiment, experiment with 300 000 cells;

FIG. 26 illustrates a number of viable tumor cells depending onconcentrations of K, Zn and Mg with natural isotope distribution;

FIG. 27 illustrates a concentration of ³⁹K, ⁶⁴Zn and ²⁴Mg (as sulphatesand chloride) which caused 50% and 100% changes in the appearance ofcells from initial tumor cells into cells of type A based on Trypan bluestaining;

FIG. 28 illustrates a comparative concentrations of K, Zn and Mgcomponents with natural isotope distribution vs. Doxorubicin EBEWE,which caused death of 50% and 100% of tumor cells obtained using trypanblue and crystal violet staining methods;

FIG. 29 illustrates characteristics of the number of nonviable tumorcells depending on the time of action of K (1), Zn (2) and Mg (3)components with natural distribution of isotopes;

FIG. 30 illustrates an ability of initial PA tumor cells to transforminto A cells in the experiment with 30 000 tumor cells after treatingthem with ³⁹K, ⁶⁴Zn and ²⁴Mg;

FIG. 31 illustrates ⁶⁴Zn concentration of 0.128 mg/ml at which about 30%cells of type A were found;

FIG. 32 illustrates 50/50% ratio of cells of types A and B at ⁶⁴Znconcentration of 3.2 mcg/ml;

FIG. 33 illustrates 100% A cells at ⁶⁴Zn concentration of 0.08 mg/ml;

FIG. 34 illustrates about 30 to 35% B Cells at ⁶⁴Zn concentration of 16mcg/ml;

FIG. 35 illustrates 100% cells of type A at ³⁹K concentration of 10mg/ml;

FIG. 36 illustrates cells of type A at the magnification of 1500 9KConcentration is 10 mg/ml;

FIG. 37 illustrates ³⁹K Effect. About 50% A Cells at ³⁹K concentrationof 2 mg/ml;

FIG. 38 illustrates a cell to B cell transformation. About 50% A Cellsat ³⁹K concentration of 2 mg/ml;

FIG. 39 illustrates 100% cells of type A at ²⁴Mg dose of 10 mg/ml;

FIG. 40 illustrates 100% cells of type A at ²⁴Mg dose of 10 mg/ml 1000Magnification;

FIG. 41 illustrates 25% cells of type A at ²⁴Mg concentration of 2mg/ml;

FIG. 42 illustrates 50% to 50% ratio of A and B types at the transitionstage ²⁴Mg dose is 10 mg/ml;

FIG. 43 illustrates 75% cells of type B at ²⁴Mg dose of 2 mg/ml;

FIG. 44 illustrates about 5% cells of type A at ²⁴Mg concentration of0.4 mg/ml;

FIG. 45 illustrates results of tests on renal cell carcinoma (PA) in arat: Adhesion and cytoskeletal proteins in renal cell carcinoma (PA)cells after their exposure to the action of light isotopes ⁶⁴Zn, ²⁴Mgand ³⁹K at doses D1 and D2;

FIGS. 46A-46B illustrate E-cadherin expression in PA cells after theaction of ⁶⁴Zn at doses D2 (FIG. 46A) and D1 (FIG. 46B)(magnification×100);

FIGS. 47A-47B illustrate N-cadherin expression in PA cells after theaction of ⁶⁴Zn at doses D2 (FIG. 47A) and D1 (FIG. 47B)(magnification×100);

FIGS. 48A-48B illustrate CD44 marker expression in PA cells after theaction of ⁶⁴Zn at doses D2 (FIG. 48A) and D1 (FIG. 48B)(magnification×100);

FIGS. 49A-49B illustrate E-cadherin expression in PA cells after theaction of ⁶⁴Zn—Z2 at doses D2 (FIG. 49A) and D1 (FIG. 49B)(magnification×100);

FIGS. 50A-50B illustrate N-cadherin expression in PA cells after theaction of ⁶⁴Zn—Z2 at doses D2 (FIG. 50A) and D1 (FIG. 50B)(magnification of ×100);

FIGS. 51A-51B illustrate CD44 marker expression in PA cells after theaction of ⁶⁴Zn—Z2 at doses D2 (FIG. 51A) and D1 (FIG. 51B)(magnification×100);

FIGS. 52A-52B illustrate E-cadherin expression in PA cells after theaction of ²⁴Mg at doses D2 (FIG. 52A) and D1 (FIG. 52B)(magnification×100);

FIGS. 53A-53B illustrate N-cadherin expression in PA cells after theaction of ²⁴Mg at doses D2 (FIG. 53A) and D1 (FIG. 53B)(magnification×100);

FIGS. 54A-54B illustrate CD44 marker expression in PA cells after theaction of ²⁴Mg at doses D2 (FIG. 54A) and D1 (FIG. 54B)(magnification×100);

FIGS. 55A-55B illustrate CD44 marker expression in PA cells after theaction of ²⁴Mg at doses D2 (FIG. 55A) and D1 (FIG. 55B)(magnification×100);

FIGS. 56A-56C illustrate E-cadherin expression in PA cells after theaction of ³⁹K at doses D2 (FIG. 56A) and D1 (FIG. 56B, FIG. 56C)(magnification×100);

FIGS. 57A-57B illustrate N-cadherin expression in PA cells after theaction of ³⁹K at doses D2 (FIG. 57A) and D1 (FIG. 57B)(magnification×100);

FIGS. 58A-58D illustrate CD44 marker expression in PA cells after theaction of ³⁹K at doses D2 (FIG. 58A) and D1 (FIG. 58B, FIG. 58C, FIG.58D) (magnification×100);

FIGS. 59A-59B illustrate E-cadherin expression in control PA cells notexposed to the action of components (magnification×100);

FIGS. 60A-60B illustrate N-cadherin expression in control PA cells notexposed to the action of components (magnification×100);

FIGS. 61A-61B illustrate CD44 marker expression in control PA cells notexposed to the action of components (magnification×100);

FIG. 62 illustrates comparative expression of E-cadherin in PA cellsafter the action of light isotopes containing materials;

FIG. 63 illustrates comparative expression of N-cadherin in PA cellsafter the action of light isotopes containing materials;

FIG. 64 illustrates comparative expression of CD44 marker in PA cellsafter the action of light isotopes containing materials (*, ** are theaverage values of various clonal expression;

FIGS. 65A-65F illustrate E-cadherin expression in control cells (notexposed to the action of preparations) on cells of A-549 (FIG. 65A, FIG.65B), MCF-7 (FIG. 65C, FIG. 65D), COLO 205 (FIG. 65E, FIG. 65F)(magnification×100);

FIGS. 66A-66F illustrate E-cadherin expression in cells after the actionof ³⁹K component: A-549 (FIG. 66A, FIG. 66B, FIG. 66C), COLO 205 (FIG.66D, FIG. 66E, FIG. 66F), (magnification×100);

FIG. 67 illustrates Adhesion and cytoskeletal proteins in the cells ofhuman non-small cell lung cancer (A-549 line) after their exposure tothe action of ³⁹K, ⁶⁴Zn and ²⁴Mg components at the dose of IC50;

FIG. 68 illustrates an adhesion and cytoskeletal proteins in the cellsof human breast cancer (MCF-7 line) after their exposure to the actionof ⁶⁴Zn and ²⁴Mg components at the dose of IC50;

FIG. 69 illustrates an adhesion and cytoskeletal proteins in the cellsof human colon adenocarcinoma (COLO 205 line) after their exposure tothe action of ³⁹K, ⁶⁴Zn and ²⁴Mg components at the dose of IC50

FIGS. 70A-70F illustrate E-cadherin expression in cells after theirexposure to the action of ⁶⁴Zn: A-549 (FIG. 70A, FIG. 70B), MCF-7 (FIG.70C, FIG. 70D), COLO 205 (FIG. 70E, FIG. 70F) (magnification×100);

FIGS. 71A-71F illustrate E-cadherin expression in cells after theirexposure to the action of ²⁴Mg: A-549 (FIG. 71A, FIG. 71B), MCF-7 (FIG.71C, FIG. 71D), COLO 205 (FIG. 71E, FIG. 71F) (magnification×100);

FIG. 72 illustrates an adhesion and cytoskeletal proteins inimmortalized cells of a rat (NRK) after their exposure to the action of⁶⁴Zn and ⁶⁴Zn—Z2 at a dose of IC50 (⁶⁴Zn—Z2 component ⁶⁴Zn after 14 daysstorage at T=+4° C.);

FIG. 73 illustrates E-cadherin expression in control cells NRK (notexposed to the action of preparations) (magnification×100); FIG. 73A andFIG. 73B illustrate E-cadherin expression in control cells NRK (notexposed to the action of preparations) (magnification×100);

FIG. 74 illustrates N-cadherin expression in control cells NRK (notexposed to the action of preparations) (magnification×100); FIG. 74A andFIG. 74B illustrate N-cadherin expression in control cells NRK (notexposed to the action of preparations) (magnification×100);

FIG. 75 illustrates CD44 expression in control cells NRK (not exposed tothe action of preparations) (magnification×100); FIG. 75A and FIG. 75Billustrate CD44 expression in control cells NRK (not exposed to theaction of preparations) (magnification×100);

FIG. 76 illustrates E-cadherin expression in NRK cells after theirexposure to the action of ⁶⁴Zn (magnification×100); FIG. 76A and FIG.76B illustrate E-cadherin expression in NRK cells after their exposureto the action of Zn (magnification×100);

FIG. 77 illustrates CD44 expression in NRK cells after their exposure tothe action of ⁶⁴Zn (magnification×100); FIG. 77A and FIG. 77B illustrateCD44 expression in NRK cells after their exposure to the action of ⁶⁴Zn(magnification×100);

FIG. 78 illustrates E-cadherin expression in NRK cells after theirexposure to the action of ⁶⁴Zn (magnification×100); FIG. 78A and FIG.78B illustrate E-cadherin expression in NRK cells after their exposureto the action of Zn (magnification×100);

FIG. 79A, FIG. FIG. 79B, and FIG. 79C illustrate results of the study oftranscription factor in human tumor cells of different genesis aftertheir exposure to the action of ³⁹K,(1) ⁶⁴Zn,(2) and ²⁴Mg(3);

FIG. 80A and FIG. 80B illustrate A-549 control (no preparations). Slugmarker expression (magnification×100);

FIG. 81A and FIG. 81B illustrate slug marker expression in cells ofA-549 line after their exposure to the action of ³⁹K at a dose of IC50(magnification×100);

FIG. 82A and FIG. 82B illustrate slug marker expression in cells ofA-549 line after their exposure to the action of ⁶⁴Zn at a dose of IC50(magnification×100);

FIG. 83A and FIG. 83B illustrate slug marker expression in cells ofA-549 line after their exposure to the action of ²⁴Mg at a dose of IC50(magnification×100);

FIG. 84A and FIG. 84B illustrate COLO 205 control (no preparations).Slug marker expression (magnification×100);

FIG. 85A and FIG. 85B illustrate slug marker expression in cells of COLO205 line after their exposure to the action of ³⁹K at a dose of IC50(magnification×100);

FIG. 86A and FIG. 86B illustrate slug marker expression in cells of COLO205 line after their exposure to the action of Zn at a dose of IC50(magnification×100);

FIG. 87A and FIG. 87B illustrate slug marker expression in cells of COLO205 line after their exposure to the action of ²⁴Mg at a dose of IC50(magnification×100);

FIG. 88A and FIG. 88B illustrate MCF-7 control (no preparations). Slugmarker expression (magnification×100);

FIG. 89A and FIG. 89B illustrate slug marker expression in cells ofMCF-7 line after their exposure to the action of ⁶⁴Zn at a dose of IC50(magnification×100

FIG. 90A, FIG. 90B, and FIG. 90C illustrate slug marker expression incells of MCF-7 line after their exposure to the action of ²⁴Mg at a doseof IC50 (magnification×100);

FIG. 91A and FIG. 91B illustrate quantitative characteristics of cellsof type A in A-549 cell line (%) after their exposure to the action of9K stored at T+4° C. for 20 days in a dissolved state in comparison withthe efficiency of a fresh solution;

FIG. 92 illustrates primary cells of A-549 line. Human lung cancerMagnification×400;

FIG. 93 illustrates an effect of ⁶⁴Zn sulphate form diluted in salinewith glucose on A-549 cells. About 20% of cells of type A after 24 hoursof the experiment Magnification×100;

FIG. 94 illustrates an effect of ⁶⁴Zn sulphate form diluted in salinewith glucose on A-549 cells. About 50% of cells of type A after 28 daysof the experiment, Magnification×100;

FIG. 95 illustrates an effect of ⁶⁴Zn sulphate form diluted in salinewith glucose on A-549 cells. About 80% of cells of type A after 60 daysof the experiment Magnification×100;

FIG. 96 illustrates an effect of ³⁹K sulphate form at a dose of 2 mg/mlon A-549 cells after 30 min following the start of the experiment. Cellsof type A are light, Magnification×400;

FIGS. 97A-97D illustrate an embryonic cells of a mouse. FIG. 97A, FIG.97B-magnification×100, C, D-magnification×400;

FIGS. 98A-98I illustrate cytogenetic characteristics: FIG. 98Aa)mitosis; FIG. 98Bh) premature chromosome condensation; FIG. 98Dd, FIG.98Ee, FIG. 98Ff) cells with micronuclei and apoptotic cells; FIG. 98Gg)nucleus with a protrusion; FIG. 98Hh, FIG. 98i ) nuclear protrusions;

FIG. 99 illustrates a level of cytogenetic characteristics in cells ofCOLO 205 line after their exposure to the action of the components;

FIG. 100 illustrates a cytogenetic characteristics in cells of A-549line after their exposure to the action of the isotopes;

FIG. 101 illustrates a cytogenetic characteristics in cells of MCF-7line after their exposure to the action of the components;

FIGS. 102A-102D illustrate the number of cellular abnormalities invarious cell lines as compared to control depending on the concentrationof the components used;

FIG. 103 illustrates quantitative characteristics of normal rat kidneycells NRK after their exposure to the action of anticancer drugDoxorubicin and ⁶⁴Zn and ³⁹K components;

FIG. 104 illustrates an appearance of initial NRK cells before the startof the experiment, Magnification×100;

FIG. 105 illustrates an effect of ⁶⁴Zn isotopes in the concentration of16 mcg/ml 30 min after the start of the experiment, Magnification×100;

FIG. 106 illustrates an effect of Zn isotopes in the concentration of 16mcg/ml and cell morphology after 48 hours in a freshly preparedsolution, Magnification×100;

FIG. 107 illustrates an effect of ⁶⁴Zn isotopes in the concentration of16 mcg/ml and cell morphology after 48 hours in a freshly preparedsolution, Magnification×400;

FIG. 108 illustrates an effect of ³⁹K isotopes in the concentration of16 mcg/ml 30 min after the start of the experiment, Magnification×100;

FIG. 109 illustrates an effect of ³⁹K isotopes in the concentration of16 mcg/ml 30 min after the start of the experiment, Magnification×400;

FIG. 110 illustrates an effect of ³⁹K isotopes in the concentration of16 mcg/ml and cell morphology after 48 hours in a freshly preparedsolution, Magnification×100;

FIG. 111 illustrates an effect of anticancer drug Doxorubicin in theconcentration of 16 mcg/ml within 30 min after the start of theexperiment, live cells are virtually absent. Magnification×100;

FIG. 112 illustrates an effect of anticancer drug Doxorubicin in theconcentration of 16 mcg/ml within 48 hours after the start of theexperiment, live cells are absent. Magnification×400;

FIGS. 113A-113E illustrate a character of migration of tumor cells ofA-549 cell line in the in vitro experiment with ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents 3 hours following the cell monolayer damage: FIG. 113A.Control group of cells (no preparations) FIG. 113B. A-549+Doxorubicin atthe dose of 2 mcg/ml, FIG. 113C. A-549+³⁹K preparation (2 mg/ml), FIG.113D. A-549+⁶⁴Zn preparation (20 mcg/ml), FIG. 113E. A-549+²⁴Mgpreparation (3 mg/ml);

FIGS. 114A-114H illustrate a character of migration of tumor cells ofA-549 cell line in the in vitro experiment with ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents 24 hours following the cell monolayer damage: FIG. 114A.Control, FIG. 114B. A-549+Doxorubicin (2 mcg/ml), FIG. 114C. A-549+³⁹Kpreparation (2 mg/ml), FIG. 114D. A-549+³⁹K preparation (3 mg/ml), FIG.114E. A-549+⁶⁴Zn preparation (20 mcg/ml), FIG. 114F. A-549+⁶⁴Znpreparation (30 mcg/ml), FIG. 114G. A-549+²⁴Mg preparation (3 mg/ml),FIG. 114H. A-549+²⁴Mg preparation (4 mg/ml);

FIGS. 115A-115H illustrate a character of migration of tumor cells ofA-549 cell line in the in vitro experiment with ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents 48 hours following the cell monolayer damage: FIG. 115A.Control, FIG. 115B. A-549+Doxorubicin (2 mcg/ml), FIG. 115C. A-549+³⁹Kpreparation (2 mg/ml), FIG. 115D. A-549+³⁹K preparation (3 mg/ml), FIG.115E. A-549+⁶⁴Zn preparation (20 mcg/ml), FIG. 115F. A-549+⁶⁴Znpreparation (30 mcg/ml), FIG. 115G. A-549+²⁴Mg preparation (3 mg/ml),FIG. 115H. A-549+²⁴Mg preparation (4 mg/ml);

FIGS. 116A-116H illustrate a character of migration of tumor cells ofA-549 cell line in the in vitro experiment with ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents 72 hours following the cell monolayer damage: FIG. 116A.Control, FIG. 116B. A-549+Doxorubicin (2 mcg/ml), FIG. 116C. A-549+³⁹Kpreparation (2 mg/ml), FIG. 116D. A-549+³⁹K preparation (3 mg/ml), FIG.116E. A-549+⁶⁴Zn preparation (20 mcg/ml), FIG. 116F. A-549+⁶⁴Znpreparation (30 mcg/ml), FIG. 116G. A-549+²⁴Mg preparation (3 mg/ml),FIG. 116H. A-549+²⁴Mg preparation (4 mg/ml);

FIGS. 117A-117G illustrate a migration activity of cells of A-549 cellline after their processing with the experimental preparation (1 hourfollowing the violation of the monolayer integrity): FIG. 117A. Control,FIG. 117B. A-549+Doxorubicin (0.2 mcg/ml), FIG. 117C. A-549+4Znpreparation (20 mcg/ml), FIG. 117D. A-549+6Zn preparation (10 mcg/ml),FIG. 117E. A-549+⁶⁴Zn preparation (20 mcg/ml)+Doxorubicin (0.02 mcg/ml),FIG. 117F. A-549+6Zn preparation (10 mcg/ml)+Doxorubicin (0.2 mcg/ml),FIG. 117G. A-549+⁶⁴Zn preparation (10 mcg/ml)+Doxorubicin (0.02 mcg/ml);

FIGS. 118A-18G illustrate a migration activity of cells of A-549 cellline after their processing with the experimental preparation (24 hoursfollowing the violation of the monolayer integrity): FIG. 118A. Control,FIG. 118B. A-549+Doxorubicin (0.2 mcg/ml), FIG. 118C. A-549+4Znpreparation (20 mcg/ml), FIG. 118D. A-549+⁶⁴Zn preparation (10 mcg/ml),FIG. 118E. A-549+⁶⁴Zn preparation (20 mcg/ml)+Doxorubicin (0.02 mcg/ml),FIG. 118F. A-549+4Zn preparation (10 mcg/ml)+Doxorubicin (0.2 mcg/ml),FIG. 118G. A-549+6Zn preparation (10 mcg/ml)+Doxorubicin (0.02 mcg/ml);

FIGS. 119A-119G illustrate a migration activity of cells of A-549 cellline after their processing with the experimental preparation (60 hoursfollowing the violation of the monolayer integrity): FIG. 119A. Control,FIG. 119B. A-549+Doxorubicin (0.2 mcg/ml), FIG. 119C. A-549+4Znpreparation (20 mcg/ml), FIG. 119D. A-549+6Zn preparation (10 mcg/ml),FIG. 119E. A-549+⁶⁴Zn preparation (20 mcg/ml)+Doxorubicin (0.02 mcg/ml),FIG. 119F. A-549+⁶⁴Zn preparation (10 mcg/ml)+Doxorubicin (0.2 mcg/ml),FIG. 119G. A-549+⁶⁴Zn preparation (10 mcg/ml)+Doxorubicin (0.02 mcg/ml);

FIGS. 120A-120G illustrate a character of the migration activity ofcells of RF cell line in the in vitro experiment with ³⁹K, 6Zn and ²⁴Mgcomponents after the first hour of observation of the cell monolayer:FIG. 120A. Control, FIG. 120B. RF+Doxorubicin, FIG. 120C. RF+³⁹Kpreparation (2 mg/ml), FIG. 120D. RF+³⁹K preparation (1 mg/ml), FIG.120E. RF+⁶⁴Zn preparation (25 mcg/ml) FIG. 120F. RF+²⁴Mg preparation (4mg/ml) FIG. 120G. RF+²⁴Mg preparation (2 mg/ml);

FIGS. 121A-121G illustrate a character of the migration activity ofcells of RF cell line in the in vitro experiment with ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents after the 24-hour observation of the cell monolayer: FIG.121A. Control, FIG. 121B. RF+Doxorubicin, FIG. 121C. RF+³⁹K preparation(2 mg/ml), FIG. 121D. RF+³⁹K preparation (1 mg/ml), FIG. 121E. RF+⁶⁴Znpreparation (25 mcg/ml) FIG. 121F. RF+²⁴Mg preparation (4 mg/ml) FIG.121G. RF+²⁴Mg preparation (2 mg/ml);

FIGS. 122A-122G illustrate a character of the migration activity ofcells of RF cell line in the in vitro experiment with ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents after the 48-hour observation of the cell monolayer: FIG.122A. Control, FIG. 122B. Effect of Doxorubicin, FIG. 122C.Fibroblasts+³⁹K preparation (2 mg/ml), FIG. 122D. RF+³⁹K preparation (1mg/ml), FIG. 122E. RF+⁶⁴Zn preparation (25 mcg/ml), FIG. 122F. RF+²⁴Mgpreparation (4 mg/ml), FIG. 122G. RF+²⁴Mg preparation (2 mg/ml);

FIGS. 123A-123G illustrate a character of the migration activity ofcells of RF cell line in the in vitro experiment with ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents after the 72-hour observation of the cell monolayer: FIG.123A. Control, FIG. 123B. Effect of Doxorubicin. FIG. 123C.Fibroblasts+³⁹K preparation (2 mg/ml), FIG. 123D. RF+³⁹K preparation (1mg/ml), FIG. 123E. RF+⁶⁴Zn preparation (25 mcg/ml) FIG. 123F. RF+²⁴Mgpreparation (4 mg/ml) FIG. 123G. RF+²⁴Mg preparation (2 mg/ml);

FIGS. 124A-124F illustrate a migration activity of cells of the RF cellline after their processing with the experimental preparation (1 hourfollowing the violation of the monolayer integrity): FIG. 124A. Control,FIG. 124B. RF+Doxorubicin (15 ng/ml), FIG. 124C. RF+Doxorubicin (5ng/ml), FIG. 124D. RF+⁶⁴Zn preparation (25 mcg/ml), FIG. 124E. RF+⁶⁴Znpreparation (25 mcg/ml)+Doxorubicin (15 ng/ml), FIG. 124F. RF+⁶⁴Znpreparation (25 mcg/ml)+Doxorubicin (5 ng/ml);

FIGS. 125A-125E illustrate a migration activity of cells of the RF cellline after their processing with the experimental preparation (24 hoursfollowing the violation of the monolayer integrity): FIG. 125A. Control,FIG. 125B. RF+Doxorubicin (15 ng/ml), FIG. 125C. RF+Doxorubicin (5ng/ml), FIG. 125D. RF+⁶⁴Zn preparation (25 mcg/ml), FIG. 125E. RF+⁶⁴Znpreparation (25 mcg/ml)+Doxorubicin (5 ng/ml);

FIGS. 126A-126E illustrate a migration activity of cells of the RF cellline after their processing with the experimental preparation (72 hoursfollowing the violation of the monolayer integrity): FIG. 126A. Control,FIG. 126B. RF+Doxorubicin (15 ng/ml), FIG. 126C. RF+Doxorubicin (5ng/ml), FIG. 126D. RF+⁶⁴Zn preparation (25 mcg/ml), FIG. 126E. RF+⁶⁴Znpreparation (25 mcg/ml)+Doxorubicin (5 ng/ml);

FIGS. 127A-127G illustrate a cell migration of the NRK cell line invitro after their processing with the experimental preparations (1 hourfollowing the violation of the monolayer integrity): FIG. 127A. Control,FIG. 127B. NRK+Doxorubicin, FIG. 127C. NRK+³⁹K component (2 mg/ml), FIG.127D. NRK+³⁹K component (1 mg/ml), FIG. 127E. NRK+⁶⁴Zn component (25mcg/ml), FIG. 127F. NRK+²⁴Mg component (4 mg/ml), FIG. 127G. NRK+²⁴Mgcomponent (2 mg/ml).

FIGS. 128A-128G illustrate a cell migration of the NRK cell line invitro after their processing with the experimental preparations (24hours following the violation of the monolayer integrity): FIG. 128A.Control, FIG. 128B. NRK+Doxorubicin, FIG. 128C. NRK+³⁹K component (2mg/ml), FIG. 128D. NRK+³⁹K component (1 mg/ml), FIG. 128E. NRK+⁶⁴Zncomponent (25 mcg/ml), FIG. 128F. NRK+²⁴Mg component (4 mg/ml), FIG.128G. NRK+²⁴Mg component (2 mg/ml);

FIGS. 129A-129G illustrate a cell migration of the NRK cell line invitro after their processing with the experimental preparations (36hours following the violation of the monolayer integrity): FIG. 129A.Control, FIG. 129B. NRK+Doxorubicin, FIG. 129C. NRK+³⁹K component (2mg/ml), FIG. 129D. NRK+³⁹K component (1 mg/ml), FIG. 129E. NRK+⁶⁴Zncomponent (25 mcg/ml), FIG. 129F. NRK+²⁴Mg component (4 mg/ml), FIG.129G. NRK+²⁴Mg component (2 mg/ml);

FIGS. 130A-130B illustrate a cell migration of the NRK cell line invitro after their processing with the experimental preparations (72hours following the scratch of the monolayer integrity): FIG. 130A.Control, FIG. 130B. NRK+Doxorubicin;

FIGS. 131A-131F illustrate a migration activity of cells of the HaCaTcell line after their processing with the experimental preparations (1hour following the violation of the monolayer integrity): FIG. 131A.Control, FIG. 131B. HaCaT+Doxorubicin (15 ng/ml), FIG. 131C.HaCaT+Doxorubicin (5 ng/ml), FIG. 131D. HaCaT+⁶⁴Zn component (25mcg/ml), FIG. 131E. HaCaT+⁶⁴Zn component (25 mcg/ml)+Doxorumbicin (15ng/ml), FIG. 131F. HaCaT+⁶⁴Zn component (25 mcg/ml)+Doxorubicin (5ng/ml);

FIGS. 132A-132F illustrate a migration activity of cells of the HaCaTcell line after their processing with the experimental preparations (24hours following the violation of the monolayer integrity): FIG. 132A.Control, FIG. 132B. HaCaT+Doxorubicin (15 ng/ml), FIG. 132C.HaCaT+Doxorubicin (5 ng/ml), FIG. 132D. HaCaT+⁶⁴Zn component (25mcg/ml), FIG. 132E. HaCaT+⁶⁴Zn component (25 mcg/ml)+Doxorubicin (15ng/ml), FIG. F. HaCaT+⁶⁴Zn component (25 mcg/ml)+Doxorubicin (5 ng/ml);

FIGS. 133A-133F illustrate a migration rate of cells of the HaCaT cellline after their processing with the experimental preparation (36 hoursfollowing the violation of the monolayer integrity): FIG. 133A. Control,FIG. 133B. HaCaT+Doxorubicin (15 ng/ml), FIG. 133C. HaCaT+Doxorubicin (5ng/ml), FIG. 133D. HaCaT+⁶⁴Zn component (25 mcg/ml), FIG. 133E.HaCaT+⁶⁴Zn component (25 mcg/ml)+Doxorumbicin (15 ng/ml), FIG. 133F E.HaCaT+⁶⁴Zn component (25 mcg/ml)+Doxorubicin (5 ng/ml);

FIGS. 134A-134I illustrate a migration activity of cells of the A-431cell line after their processing with the experimental preparation (1hour following the violation of the monolayer integrity): FIG. 134A.Control, FIG. 134B. A-431+Doxorubicin (0.1 mcg/ml), FIG. 134C.A-431+Doxorubicin (0.02 mcg/ml), FIG. 134D. A-431+⁶⁴Zn component (20mcg/ml), FIG. 134E. A-431+⁶⁴Zn component (10 mcg/ml), FIG. 134F.A-431+⁶⁴Zn component (20 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 134G.A-431+⁶⁴Zn component (10 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 134H.A-431+⁶⁴Zn component (20 mcg/ml)+Doxorubicin (0.02 mcg/ml), FIG. 134I.A-431+⁶⁴Zn component (10 mcg/ml)+Doxorubicin (0.02 mcg/ml);

FIGS. 135A-135I illustrate a migration activity of cells of the A-431cell line after their processing with the experimental preparation (24hours following the violation of the monolayer integrity): FIG. 135A.Control, FIG. 135B. A-431+Doxorubicin (0.1 mcg/ml), FIG. 135C.A-431+Doxorubicin (0.02 mcg/ml), FIG. 135D. A-431+⁶⁴Zn component (20mcg/ml), FIG. 135E. A-431+⁶⁴Zn component (10 mcg/ml), FIG. 135F.A-431+⁶⁴Zn component (20 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 135G.A-431+⁶⁴Zn component (10 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 135H.A-431+⁶⁴Zn component (20 mcg/ml)+Doxorubicin (0.02 mcg/ml), FIG. 135I.A-431+⁶⁴Zn component (10 mcg/ml)+Doxorubicin (0.02 mcg/ml);

FIGS. 136A-136I illustrate a migration activity of cells of the A-431cell line after their processing with the experimental preparation (48hours following the violation of the monolayer integrity): FIG. 136A.Control, FIG. 136B. A-431+Doxorubicin (0.1 mcg/ml), FIG. 136C.A-431+Doxorubicin (0.02 mcg/ml), FIG. 136D. A-431+⁶⁴Zn component (20mcg/ml), FIG. 136E. A-431+⁶⁴Zn component (10 mcg/ml), FIG. 136F.A-431+⁶⁴Zn component (20 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 136G.A-431+⁶⁴Zn component (10 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 136H.A-431+⁶⁴Zn component (20 mcg/ml)+Doxorubicin (0.02 mcg/ml), FIG. 136I.A-431+⁶⁴Zn component (10 mcg/ml)+Doxorubicin (0.02 mcg/ml);

FIGS. 137A-137B illustrate a migration of cells of the A-431 cell lineafter their processing with Doxorubicin at a dose of 0.1 mcg/ml: FIG.137A. in 40 hours after the start of the experiment, FIG. 137B. in 45hours after the start of the experiment;

FIGS. 138A-138B illustrate a migration of cells of the A-431 cell lineafter their processing with the experimental preparation (72 hoursfollowing the violation of the monolayer integrity): FIG. 138A.A-431+⁶⁴Zn component (20 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 138B.A-431+⁶⁴Zn component (10 mcg/ml)+Doxorubicin (0.1 mcg/ml);

FIGS. 139A-139F illustrate a migration activity of cells of the MM-4cell line in vitro after their processing with the experimentalpreparation (3 hours following the violation of the monolayerintegrity): FIG. 139A. Control, FIG. 139B. MM-4+Doxorubicin (0.1mcg/ml), FIG. 139C. MM-4+Doxorubicin (0.01 mcg/ml), FIG. 139D. MM-4+⁶⁴Zncomponent (15 mcg/ml), FIG. 139E. MM-4+⁶⁴Zn component (15mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 139F. MM-4+⁶⁴Zn component (15mcg/ml)+Doxorubicin (0.01 mcg/ml);

FIGS. 140A-140F illustrate a migration activity of cells of the MM-4cell line in vitro after their processing with the experimentalpreparation (24 hours following the violation of the monolayerintegrity): FIG. 140A. Control, FIG. 140B. MM-4+Doxorubicin (0.1mcg/ml), FIG. 140C. MM-4+Doxorubicin (0.01 mcg/ml), FIG. 140D. MM-4+⁶⁴Zncomponent (15 mcg/ml), FIG. 140E. MM-4+⁶⁴Zn component (15mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 140F. MM-4+⁶⁴Zn component (15mcg/ml)+Doxorubicin (0.01 mcg/ml);

FIGS. 141A-141F illustrate a migration activity of cells of the MM-4cell line in vitro after their processing with the experimentalpreparation (48 hours following the violation of the monolayerintegrity): FIG. 141A. Control, FIG. 141B. MM-4+Doxorubicin (0.1mcg/ml), FIG. 141C. MM-4+Doxorubicin (0.01 mcg/ml), FIG. 141D. MM-4+⁶⁴Zncomponent (15 mcg/ml), FIG. 141E. MM-4+⁶⁴Zn component (15mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG. 141F. MM-4+⁶⁴Zn component (15mcg/ml)+Doxorubicin (0.01 mcg/ml);

FIGS. 142A-142C illustrate a restoration of cell monolayer of the MM-4cell line in the in vitro experiment in 70 hours after they wereprocessed with the experimental components, FIG. 142A. Control, FIG.142B. MM-4+⁶⁴Zn component (15 mcg/ml)+Doxorubicin (0.1 mcg/ml), FIG.142C. MM-4+⁶⁴Zn component (15 mcg/ml)+Doxorubicin (0.01 mcg/ml);

FIGS. 143A-143B illustrate a restoration of cell monolayer of the MM-4cell line in the in vitro experiment in 72 hours after they wereprocessed with the experimental components FIG. 143A. Control, FIG.143B. MM-4+⁶⁴Zn component (15 mcg/ml);

FIGS. 144A-144B present a table setting forth the characteristics ofeffects of light isotope containing materials, control group of cellsand Doxorubicin in the experiment using the scratch assay migrations andthe character of the combined effects of doxorubicin and light isotopes;

FIG. 145 illustrates a mass spectrum representing recorded m/zrelationship graphically;

FIG. 146 illustrates a method of direct imaging of isotope K-39 on thesample surface;

FIG. 147 illustrates a view of homogeneity of potassium isotopedistribution obtained by producing a profilogram;

FIGS. 148A-148C illustrate an isotope distribution in young and oldbiological tissue;

FIG. 149 illustrates a mass spectrum of the young tissue sample in arange of 1 to 50 amu;

FIG. 150 illustrates a mass spectrum of the old tissue sample in a rangeof 1 to 50 amu;

FIG. 151 illustrates a mass spectrum of the young tissue sample in arange of 50 to 100 amu;

FIG. 152 illustrates a mass spectrum of the old tissue sample in a rangeof 50 to 100 amu;

FIG. 153 illustrates a diagram of deviation of the isotopic compositionof young and old tissues from the natural isotope distribution;

FIG. 154 illustrates a comparative assessment of changes in theconcentration of heavy and light isotopes in biological tissues ofdifferent ages;

FIGS. 155A-155C illustrate an isotope distribution in samples 12 and 14;

FIGS. 156A-156D illustrate an isotope distribution in samples 18 and 20;

FIG. 157 illustrates a diagram of deviations of the isotopic compositionin normal and pathological tissues of an adult from the natural isotopedistribution obtained as a result of analysis of samples 12 and 14;

FIG. 158 illustrates a comparative assessment of changes in theconcentration of heavy isotopes in samples 12 and 14;

FIG. 159 illustrates a diagram of deviations of the isotopic compositionin normal and pathological tissues of an adult from the natural isotopedistribution obtained as a result of analysis of samples 18 and 20;

FIG. 160 illustrates a comparative assessment of changes in theconcentration of heavy isotopes in samples 18 and 20;

FIGS. 161A-161D illustrate a quantification of isotope content in thesamples of fungus and cortex and comparison of the obtained results withnatural distribution of isotopes;

FIG. 162 illustrates a diagram of deviation of the isotopic compositionin the samples of fungus and cortex from the natural isotopedistribution;

FIG. 163 presents data on the effect of the deuterium-depleted solutioncomprising ⁶⁴Zn_(e) aspartate on survival of experimental animals (modelL1210);

FIG. 164 illustrates N-cadherin expression in NRK cells after theirexposure to the action of ⁶⁴Zn (magnification×100); FIG. 164A and FIG.164B illustrate N-cadherin expression in NRK cells after their exposureto the action of ⁶⁴Zn (magnification×100);

FIG. 165A, FIG. 165B, and FIG. 165C illustrate CD44 expression in NRKcells after their exposure to the action of Zn (magnification×100).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specifically stated, all scientific and technical termsused herein have the same meaning as commonly understood by a person ofordinary skill in the art.

The term “isotope”, as used herein, refers to a variant of a particularchemical element which are rather similar in their physical and chemicalproperties but have a different atomic mass. According to theproton-neutron model developed by D.I. Ivanenko and W. Heisenberg(1932), atoms of all chemical elements consist of three types ofelementary particles: positively charged protons, negatively chargedelectrons, and neutrons that have no charge. The number of protons p inthe nucleus determines the atomic number Z of the chemical element inMendeleev's periodic table. The proton and the neutron, which have acommon name—nucleons—have almost identical weight. The mass of theneutron (1.00866 amu) is somewhat greater than the proton mass (1.00727amu). The electron mass is much smaller than that of the nucleons (forexample, the proton-to-electron mass ratio is 1836.13). Therefore, themass of the atom is concentrated in its nucleus. Hence, the mass numberof the atom A is connected with the atomic number by a simple relationA=p+n=Z+n, where n is the number of neutrons in the nucleus of an atom.The number of protons in the nucleus of an atom uniquely determines theposition of an element in the periodic table of the elements.Furthermore, the number of protons determines the number of electronspresent in a neutral atom thus determining the chemical properties ofthis atom. However, atoms with the same atomic number Z (and hence thenumber of protons p) may have different neutron numbers n. Thus atomswith different atomic mass numbers may occupy the same position on theperiodic table. Chemical elements having the same atomic number but adifferent atomic mass are known as isotopes.

The “light isotopes” of interest for the present invention include K-39,Mg-24, Zn-64, Rb-85, Si-28, Ca-40, Cu-63, Fe-54, Cr-52, Ni-58, Mo-92,Se-74, Br-79, and Cl-35.

The “natural abundance” of an isotope refers to the fraction of thetotal amount of the corresponding element that the isotope represents,on a mole-fraction basis (that is, not, for example, on a mass basis).For example, if ⁶⁴Zn had an earth natural abundance of 48.63%, thatwould mean that 48.63% of Zn atoms on earth are the isotope ⁶⁴Zn. When acomposition is “enriched” for a certain isotope, the abundance of theisotope in the composition is greater than the isotope's naturalabundance. For the preceding ⁶⁴Zn example, a composition in which ⁶⁴Znconstitutes more than 48.63% of the total Zn in the composition, on amole-fraction basis, would be “enriched” for ⁶⁴Zn. Throughout thisapplication, a subscript “e” following a light isotope chemical symbolor element name indicates that the designated element is enriched forthat isotope. For example, ⁶⁴Zn refers to the light isotope zinc-64,whereas ⁶⁴Zn_(e) refers to zinc that is enriched for zinc-64. Thus,“⁶⁴Zn_(e) aspartate,” for example, refers to zinc aspartate in which thezinc is enriched for zinc-64.

The proportion of an element that is present as a particular isotope ofthe element is often expressed relative to a ratio called the standardisotope ratio or SIR. The abundance of the isotope of interest is thenumerator of the SIR and the abundance of the most abundant isotope isthe denominator. For example, ¹²C is the most abundant carbon isotopeand ¹³C is a second carbon isotope. Assuming a standard abundance valuefor C-12 of 98.89% and a standard abundance value for ¹³C of 1.11%, theSIR for ¹³C would be 1.11/98.89=0.01122. Each SIR is obtained from areference material. Deviations from the SIR may be observed innon-reference materials.

For ease and convenience, the abundance of a heavy isotope in a materialof interest may be expressed relative to the heavy isotope's “standard”abundance in the reference material by reference to the difference inisotope ratios, expressed in parts per thousand or “%” and referred toas delta-[isotope] or δ−[X], where “[X]” represents the isotope ofinterest. The δ value is calculated as ((R_(sample)−SIR)/SIR)×1000%,equivalent to ((R_(sample)/SIR)−1)×1000%, where R_(sample) is theisotope ratio of the sample under evaluation. For example, if the carbonstandard contains 99% ¹²C and 1% ¹³C, and the sample has 98.95% ¹²C and1.05% ¹³C, then the corresponding SIR, or ¹³C/¹²C of the standard, is1/99, or 0.0101, and the ¹³C/¹²C of the sample is 1.05/98.95, or 0.0106,so δ¹³C_(sample)=((0.0106/0.0101)−1)×1000%=49.5% (also known as 49.5permil) or 0.0495.

Relative abundance of an isotope can also be expressed with respect todifferent isotopes' absolute abundances expressed in terms of “atompercent” and “fractional abundance.” Atom percent is calculated as(^(A)X/(sum of all X isotopes))×100, whereas fractional abundance issimply ^(A)X/(sum of all X isotopes), where “^(A)X” is a measure of thequantity of isotope A of element X in a sample, and “sum of all Xisotopes” is a measure of the total quantity of element X in a sample.Enrichment for a specific isotope in a sample of interest may beexpressed as a percentage of the fractional abundance or atom percent ofa reference standard. For example, if a reference standard containedpotassium, of which 93.3% was ³⁹K, then the atom percent of ³⁹K would be93.3% and its fractional abundance would be 0.933. If a sample were tocontain potassium, of which 95.0% was ³⁹K, then the sample would beenriched with respect to ³⁹K by (95.0-93.3)/93.3=1.82%. If a sample weresaid to be enriched with respect to ³⁹K by 5% relative to the standard,then the percentage of the potassium in the sample that is ³⁹K would be1.05×93.3=97.97%.

The degree of enrichment of a certain isotope also may be expressed withrespect to the difference D(I) (where “I” represents the identity of theisotope) between 100% and the isotope's natural abundance, expressed asa mole percentage of the total amount of the corresponding element. Forexample, if ⁶⁴Zn had a natural abundance of 48.63%, thenD(⁶⁴Zn)=100%−48.63%=51.37%. A sample's enrichment may then be expressedas the amount by which D is reduced. For the ⁶⁴Zn example, for a samplein which D(⁶⁴Zn) is reduced by 10%, D(⁶⁴Zn) would equal 51.37% minus(10%×51.37), which equals 46.233%, and the ⁶⁴Zn atom percent in thesample would be (100%−46.233%), which equals 53.767%. The sample wouldthus be characterized as enriched for ⁶⁴Zn by 10% of D.

The authors of the present invention have discovered that a compositionthat comprises at least one light isotope selected from the groupconsisting of K-39, Mg-24, Zn-64, Rb-85, Si-28, Ca-40, Cu-63, Fe-54,Cr-52, Ni-58, Mo-92, Se-74, Br-79, and Cl-35, wherein the composition isenriched for the at least one light isotope relative to the naturalabundance of the isotope, has pronounced therapeutic effects, as furtherdescribed below. In addition to being enriched for a light isotope asdescribed above, the composition may comprise one or more additionalactive ingredients, as well as water and inert auxiliary ingredientssuch as carriers, diluents and the like which are used to formulate thesaid composition and may be pharmaceutically acceptable orpharmaceutically unacceptable (which are used as intermediates in thepreparation of pharmaceutically acceptable agents).

As used herein, the terms “treat,” “treating,” “treatment of” acondition encompass performing an act (such as administering thecomposition of the invention) in order to cure, eradicate, or diminishthe severity of, the condition treated. These terms thus encompassaccomplishing any one or more of curing, eradicating, and diminishingthe severity of the condition treated. As used herein, the terms“prevent,” “preventing,” “prevention of” a condition encompassperforming an act (such as administering the composition of theinvention) in order to prevent the occurrence of the condition anddiminish the severity of the condition if it occurs subsequent to theact. These terms thus encompass accomplishing any one or more of whollypreventing the condition from occurring and diminishing the severity ofthe condition if it occurs subsequent to the act.

For reference with respect to the invention, the above-listed isotopesare considered to have the natural abundances, on a mole-fraction basis,shown in the following table. The table also shows the correspondingpercentages preferred for use in the compositions of the invention, on amole-fraction basis (lower limits are provided; in every case, themaximum theoretical upper limit is 100%). For example, in a compositionof the invention that uses a therapeutic amount of ⁶⁴Zn, the zinc in thecomposition preferably would contain at least about 90% ⁶⁴Zn.Compositions that contain isotopes with lower levels of enrichment mayalso be effective and are within the scope of the invention.

Isotope Natural abundance (%) % for therapeutic use ³⁹K 93.2581 at leastabout 98% ²⁴Mg 78.99  at least about 95%* ⁶⁴Zn 48.63  at least about90%* ⁸⁵Rb 72.17  at least about 90%* ²⁸Si 92.2297 at least about 95%⁴⁰Ca 96.94 at least about 98% ⁶³Cu 69.17  at least about 90%* ⁵⁴Fe 5.845 at least about 80%* ⁵²Cr 83.789 at least about 90% ⁵⁸Ni 68.0769  atleast about 90%* ⁹²Mo 14.84  at least about 80%* 74Se 0.89  at leastabout 50%* ⁷⁹Br 50.69  at least about 90%* ³⁵Cl 75.78  at least about90%* *In some embodiments, an enrichment level about 10 percentagepoints lower may be used for this isotope for therapeutic applicationand preferably for prophylactic use. For example, for ⁶⁴Zn, acomposition in which the zinc contains at least about 80% ⁶⁴Zn may beadministered for therapeutic purposes.

In a therapeutic composition of the invention, the composition isenriched for at least one light isotope selected from the group thatincludes ³⁹K, ²⁴Mg, ⁶⁴Zn, ⁸⁵Rb, ²⁸Si, ⁴⁰Ca, ⁶³Cu, ⁵⁴Fe, ⁵²Cr, ⁵⁸Ni,⁹²Mo, 74Se, ⁷⁹Br, and ³⁵Cl, or any combination thereof. At least onelight isotope may be present as a component of a chemical compound, suchas the salt of an organic or inorganic acid, which is pharmaceuticallyacceptable and can be administered to humans and veterinary animals(such as veterinary mammals). Exemplary salts include the chloride,citrate, sulfate, aspartate, glutamate, asparaginate and ethylenediamine disuccinic acid (referred to herein interchangeably as “EDDS”and “EDDA”) salts of the light isotope, and hydrates of such salts. Forexample, zinc enriched for ⁶⁴Zn may be present in the form of the saltzinc aspartate or the salt zinc asparaginate.

The therapeutic composition of the invention may be prepared by making acompound that is enriched for a light isotope, such as the salt of anorganic or inorganic acid and the light isotope, purifying the obtainedcompound by standard methods, and subsequent preparation of thecomposition of the invention in any appropriate form, such as an aqueoussolution. Such methods are well known and the person of ordinary skillin the art can prepare a compound containing a light isotope of aparticular chemical element, its salt in particular. The preparationprocess of the complex of aspartic acid and zinc which is enriched forthe isotope ⁶⁴Zn is described in an Example below. The lightisotope-enriched compound may be administered as a component oringredient of any convenient dosage form. Such dosage forms includetopical dosage forms such as solutions, sprays, lotions, salves,ointments, gels, creams, soaps, shampoos, and foams, oral dosage formssuch as tablets, capsules, syrups, suspensions, lozenges, gums, sprays,patches, and solutions, injection dosage forms such as solutions, e.g.aqueous solutions, and conventional dosage forms suitable for otherconventional routes of administration. Conventional dosage forms arewell-known to the person of ordinary skill in the art. Examples of suchdosage forms and their preparation are described in, for example, LoydV. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems (8th ed. 2005) (Lippincott Williams & Wilkins), andpublications cited therein.

The therapeutic composition of the invention may contain water assolvent. The composition of the invention may be in the form of anaqueous solution to be administered by any suitable route, such asorally and topically, or in the form of a gel, salve, ointment, paste,cream, foam, lotion, drops, or other topical composition. In a preferredaqueous solution, the water used is enriched for ¹⁶O and enriched for ¹Hby being depleted for ²H. The composition may further include anysuitable excipient known to the person of ordinary skill in the art,including solvents, binders, lubricants, emulsifiers, detergents,surfactants, buffers, stabilizers, and preservatives. These aredescribed in commonly used references, such as the Handbook ofPharmaceutical Excipients.

The concentration of the light isotope-enriched element in a compositionof the invention, relative to the total weight of the composition,varies according to conventional composition weights and the dosage ofthe light isotope-enriched element. Appropriate dosages of the lightisotope-enriched element are set forth below. Preferably the compositionof the invention comprises an effective amount of at least one lightisotope, wherein “effective amount” refers to that amount that providesa therapeutic effect such as an anticancer effect. As stated above, thequantity of light isotope that is effective is proportional to thequantity of the corresponding element that is present in the body. Wherethe body contains a relatively large quantity of the element, acorrespondingly relatively large amount of the element's light isotopewill be required to provide an effective dosage amount. On the otherhand, where the body contains a relatively small quantity of theelement, a correspondingly relatively small amount of the element'slight isotope will be required to provide an effective dosage amount.These quantities are reflected in the “guidance amounts” for eachelement, the recommended amount for daily human consumption, as detailedbelow.

In certain embodiments, the preferred dosage of any of the lightisotopes is proportional to various authoritative daily ingestionguidances (e.g. recommended dietary allowance (USRDA), adequate intake(AI), recommended dietary intake (RDI)) of the corresponding element.The light isotope dosage is preferably between about ½ and about 30times the guidance amount of the corresponding element, more preferablybetween about 1 and about 10 times the guidance amount, even morepreferably between about 1 and about 3 times the guidance amount.Generally, the low end of the dose range to be administered daily isabout ½ the guidance daily amount, whereas the high end is as follows:total daily oral doses can be as high as about 30 times the guidancedaily amount, total daily doses administered by intraperitonealinjection can be as high as about 20 times the guidance daily amount,and total daily doses administered intravenously can be as high as about7 times the guidance daily amount.

Thus, in preferred embodiments, a single dose of a composition of theinvention for daily administration would be formulated to comprise aquantity within these ranges, such as about ½, about 1, about 3, about5, about 10, and about 20 times the guidance amount. These amountsgenerally are for oral intake or topical application. In someembodiments, the preferred intravenous dosage is lower, such as fromabout 1/10 to about ½ the guidance amount. Doses at the low end of theseranges are appropriate for anyone with a heightened sensitivity to aspecific element or class of elements (e.g., those with kidneyproblems). For zinc, the guidance amount ranges from 2 mg in infants to8-11 mg (depending on sex) for ages 9 and up. Guidance amounts for someof the elements used in the compositions of the invention are presentedbelow based on information obtained fromhttps://ods.od.nih.gov/factsheets/list-all/ andhttps://health.gov/dietaryguidelines/2015/guidelines/appendix-7/,summarized below. Daily dosages discussed throughout this applicationmay be subdivided into fractional dosages and the fractional dosagesadministered the appropriate number of times per day to provide thetotal daily dosage amount (e.g. ½ the daily dose administered twicedaily, ⅓ the daily dose administered three times daily, etc.).

Element/Isotope guidance amount, daily magnesium/ 30-420 mg ²⁴Mg(400-420 mg in males 14+; 310-360 mg in females 14+) potassium/ 1 to 3years: 3 g/day ³⁹K 4 to 8 years: 3.8 g/day 9 to 13 years: 4.5 g/day 14to 18 years: 4.7 g/day Age 19 and older: 4.7 g/day chromium/ Hexavalentchromium should be avoided. ⁵²Cr Chromium complexes are preferred fororal administration (e.g. picolinate, dinico- cysteinate, as nicotinicacid complex). For parenteral administration, chromic chloride at 4mcg/ml may be used. 0-6 mos. 0.2 mcg 7-12 mos. 5.5 mcg 1-3 yrs 11 mcg4-8 yrs 15 mcg 9-13 yrs females: 21 mcg, males: 25 mcg 14-18 yrsfemales: 24 mcg, males; 35 mcg 19-50 yrs females: 25 mcg, males: 35mcg >50 yrs females: 20 mcg, males: 30 mcg Iron/ Birth to 6 months 0.27mg ⁵⁴Fe 7-12 months 11 mg 1-3 years 7 mg 4-8 years 10 mg 9-13 years 8 mg14-18 years males: 11 mg, females: 15 mg 19-50 years males: 8 mg,females: 18 mg Adults 51 years and older 8 mg Copper/ adequate: ⁶³Cu 0to 6 months: 200 mcg 7 to 12 months: 220 mcg recommended: 1 to 3 years:340 mcg 4 to 8 years: 440 mcg 9 to 13 years: 700 mcg 14 to 18 years: 890mcg 19 and older: 900 mcg Zinc/ Birth to 6 months 2 mg ⁶⁴Zn 7 months-3years 3 mg Children 4-8 years 5 mg Children 9-13 years 8 mg 14-18 years(boys) 11 mg 14-18 years (girls) 9 mg Adults (men) 11 mg Adults (women)8 mg Calcium/ 1-3 years 700 mg ⁴⁰Ca 4-8 years 1000 mg 9 years-adult 1300mg Chlorine/ 0-6 mos. 180 mg ³⁵Cl 6-12 mos. 570 mg 1-10 yrs. 1.75 g11-18 yrs. 2.2 g 19-50 yrs. 2.3 g over 50 yrs. 1.9 g Selenium/ Birth to6 months 15 mcg ⁷⁴Se 7 months-3 years 20 mcg Children 4-8 years 30 mcgChildren 9-13 years 40 mcg 14 years and older 55 mcg

For purposes of the invention, for the following substances, thefollowing amounts are considered to be benchmark daily intakes (guidanceamounts): rubidium: between about 1 and 2 mg per day; silicon: about 10mg; molybdenum: about 1.5 mg; nickel: about 100 mcg; bromine: 1 mg/kgbody mass. Thus, a composition of the invention that contains lightrubidium, for example, preferably contains ⁸⁵Rb_(e) in an amount betweenabout 1 times and about 20 times these amounts (between about 1 mg andabout 40 mg), more preferably between about 1 and about 10 times theseamounts, and even more preferably between about 1 and about 3 timesthese amounts. (Throughout the application, the term “mcg” has itsconventional meaning of “microgram(s)”.) Based on the above, in certainembodiments, a composition of the invention containing ⁶⁴Zn_(e) as theactive ingredient, prepared for administration to a male 19 years of ageor older, preferably contains, in a single dose, between about 11 mg andabout 220 mg ⁶⁴Zn_(e) (zinc enriched for ⁶⁴Zn), more preferably betweenabout 11 mg and about 110 mg ⁶⁴Zn_(e), even more preferably betweenabout 11 mg and about 33 mg ⁶⁴Zn_(e). Such a composition may be, forexample, for oral administration, such as a tablet or capsule, or fortopical administration, such as a cream, gel, ointment, or lotion(optionally containing DMSO or other absorption-enhancing agent andother appropriate excipients).

In certain preferred embodiments, the daily dosages of ⁶⁴Zn_(e) in acomposition of the invention, such as a tablet, capsule, salve, cream,lotion, or ointment, comprise between about 10 and about 50 mg of ⁶⁴Zn,such as about 15 mg, about 30 mg, or about 45 mg of ⁶⁴Zn_(e), which maybe elemental or in the form of ⁶⁴Zn_(e) asparaginate, ⁶⁴Zn_(e)aspartate, or another pharmaceutically acceptable ⁶⁴Zn_(e) salt orcomplex. Such compositions preferably contain, in addition to the⁶⁴Zn_(e) compound, excipients suitable to the formulation type. Inanalogous preferred embodiments, the daily dosages of another lightisotope may be determined relative to these dosages and the relativeguidance amounts of ⁶⁴Zn_(e) and the other light isotope. For example,if the guidance amount of another light isotope were one-half (½) thatof zinc, then preferred daily doses of the other light isotope in acomposition of the invention would be between about 5 mg and about 25mg, such as about 7.5 mg, about 15 mg, or about 22.5 mg, in elementalform or as a pharmaceutically acceptable salt or complex.

In some embodiments, a composition of the invention may contain two ormore compounds that are each enriched for a light isotope. Thepercentages and masses above may represent each of the lightisotope-enriched compounds and may alternatively represent their totalpercentage or mass.

The composition of the invention may include an additional active agent,as well as auxiliary agents which improve the stability and therapeuticproperties of the composition and are generally present in many finishedpharmaceutical products.

Compositions that contain zinc are known and include topicalformulations that contain 20% or 40% w/w zinc oxide and oralformulations such as tablets and capsules that contain 30 mg or 50 mgzinc in various forms. In an embodiment, the present invention providescomparable compositions in which the zinc is enriched for ⁶⁴Zn. Forexample, the zinc in such compositions may contain at least about 90%⁶⁴Zn, such as between about 90% and about 99.9% ⁶⁴Zn, such as about 90%,about 95%, about 99%, or about 99.9% ⁶⁴Zn, on a mole fraction basis.Examples of such compositions include: a paste that contains betweenabout 20% w/w and about 40% w/w ⁶⁴Zn_(e) oxide, such as about 20%, about30%, or about 40% w/w ⁶⁴Zn_(e) oxide; an ointment that contains about20% w/w ⁶⁴Zn_(e) oxide; tablets and capsules that contain between about30 mg and about 50 mg of ⁶⁴Zn_(e), such as about 30, about 40, or about50 mg ⁶⁴Zn_(e), present in the form of zinc gluconate, zinc bisglycinatechelate, or any pharmaceutically acceptable zinc salt such as thoseenumerated above (aspartate, asparaginate, glutamate, EDDA, etc.). Suchcompositions preferably also contain excipients suitable to eachformulation type. Examples of such excipients and representative paste,ointment, tablet and capsule compositions, and their preparation, aredisclosed, for example, in Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems (8th ed. 2005) (Lippincott Williams & Wilkins)(capsules and tablets are discussed, for example, at pages 204-75, andointments and pastes are discussed, for example, at pages 276-97; bothsections are incorporated by reference herein in their entirety), andpublications cited therein. Dosage amounts of any topical compositionsof the invention preferably vary with skin thickness at the site ofadministration, with higher dosage amounts being used on thicker skinand lower dosages on thinner skin.

The pH of the said composition, when aqueous (including emulsions suchas oil-in-water and water-in-oil emulsions), may be between about 2 andabout 10, such as between about 2 and about 4, between about 4 and about6, between about 6 and about 8, and between about 8 and about 10. Forexample, the composition may have a pH of about 2, about 3, about 4,about 5, about 6, about 7, or about 8, as appropriate for the route ofadministration and site of administration.

Referring to FIG. 1, a pharmaceutical composition of the presentinvention is generally shown at 10. The pharmaceutical composition 10 isused for improving health, cure abnormalities and degenerative disease;achieve anti-aging effect of therapy and therapeutic effect on mammals,such as, for example, a human 12. The pharmaceutical composition 10includes a pharmaceutical carrier 14 and an isotope selective ingredient16 including at least one of a chemical element 18 and a chemicalcompound 20 containing the chemical element whereby isotope distributionin the at least one of the chemical element 18 and the chemical compound20 containing the chemical element 18 is different from naturaldistribution of at least one of isotopes wherein the part of selectedisotope of the chemical element 18 ranges from 0 to 100%.

As discussed above, the selected isotopes include at least one of K-39;Mg-24; Zn-64; Rb-85; Si-28; Ca-40; Cu-63; Fe-54; Cr-52; Ni-58; Mo-92;Se-74; Br-79; Cl-35 and combinations thereof. The pharmaceutical of thecarrier pharmaceutical composition is used in the form of a solution, agel, a cream, a spray, an aerosol, a patch, nanoparticles, inorganicmolecules, organic molecules, a plant, a fruit and a vegetable. Thepharmaceutical composition 10 includes combination of at least two ofthe isotopes wherein one of the isotopes is lighter in weight than theother of the isotopes to achieve therapeutic effect. The light isotopesof the pharmaceutical composition are K-39; Mg-24; Zn-64; Rb-85; Si-28;Ca-40; Cu-63; Fe-54; Cr-52 Ni-58; Mo-92; Se-74; Br-79; (1-35. Thechemical compounds 20 of the pharmaceutical composition 10 include theisotopes such as at least one of oxides, sulfates, citrates gluconate,and a chelate containing a ligand bonded to a central metal atom atleast two points. The chemical elements 18 and the chemical compounds 20are food supplements.

A method of using the pharmaceutical composition to improve health, cureabnormalities and degenerative disease and achieve therapeutic effect onmammals is provided. The method begins with preparing the pharmaceuticalcarrier and the isotope selective ingredient including at least one ofthe chemical element and the chemical compound containing the chemicalelement whereby isotope distribution in the at least one of the chemicalelement and the chemical compound containing the chemical element isdifferent from natural distribution of at least one of isotopes whereinthe part of selected isotope of the chemical element ranges from 0 to100%.

The next step of the method includes administering the first of theisotopes at least prior to and after surgical removal of a solid tumorto prevent possible metastases and occurrence of secondary effects andto prevent metastasizing followed by administering a second of theisotopes to transform a cancer cell phenotype into a normal cell. Thefirst of the isotopes administered prior to and after surgical removalof the solid tumor to prevent possible metastases include at least oneof K-39, Mg-24, Zn-64, Rb-85, Si-28 and combination thereof. The secondof the isotopes used to transform the cancer cell phenotype into thenormal cell includes at least one of K-39, Mg-24, Zn-64, Rb-85, Si-28and combination thereof. The step of administering the pharmaceuticalcarrier can be carried orally, intravenously and locally withoutlimiting the scope of the present invention. The isotope selectiveingredient may be administered prior to and after surgical removal of asolid tumor to prevent further spreading of cancer cells andmetastasizing of a primary tumor.

The isotope selective ingredient is administered at least prior to,after and simultaneously with chemotherapeutic agent are used to amplifytherapeutic effect on cancer tissue and to protect healthy tissue fromchemotherapy side effects and from immunotherapy side effect. Thechemotherapeutic agent includes at least one of Actinomycin, All-transretinoic acid, Azacitidine, Azathioprine, Bleomycin, Bortezomib,Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide,Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine,Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel,Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine,Vincristine, Vindesine, and Vinorelbine. The method of the presentinvention allows administering the isotope selective ingredient to causefast and significant reduction in degree of malignancy and to inducechanges of cells phenotype form malignant phenotype to a benign ornormal phenotype.

Numerous comprehensive Assessment of Antitumor Activity of ³⁹K, ⁶⁴Zn and²⁴Mg in In Vitro and In Vivo Experiments were conducted pertaining topreparation and administration of the inventive the pharmaceuticalcomposition. Such experiments and tests will be presented and describedherebelow as Example 1, Leukemia Cell Lines, Burkitt lymphoma (Namalwaline) and acute promyelocyte leukemia (HL-60 line) cells under theaction of components containing ³⁹K, ⁶⁴Zn and ²⁴Mg and Phenotypicfeatures of acute promyelocyte leukemia cells (HL-60 line) under theaction of components containing ³⁹K, ⁶⁴Zn and ²⁴Mg, Example 2, Cells ofRenal Carcinoma in a Rat (PA), Antitumor and biological activity ofcomponents containing ⁶⁴Zn and ²⁴Mg and Zn, Mg, K elements on a model ofrenal carcinoma (PA) in a rat in the in vitro experiment,Immunocytochemical study of adhesion proteins, cytoskeleton (cadherins)and CD 44 stem cell marker after their exposure to the action ofcomponents containing ³⁹, ⁶⁴Zn and ²⁴Mg on a model of renal cellcarcinoma (PA) in a rat, Detection of cumulative properties of ⁶⁴Zn,Example 3, Phenotype and cytogenetic characteristics of A-549, MCF-7 andCOLO 205 human tumor cell lines. Cumulative effect of anti-tumorproperties of light isotopes, Epithelial-mesenchymal transition and atranscription factor as criteria for assessing phenotype in A-549, MCF-7and COLO 205 human tumor cells and normal rat cells after their exposureto the action of components containing ³⁹K, ⁶⁴Zn and ²⁴Mg. Detection ofthe effect of cumulation of antitumor properties in the group ofsubstances under study on the A-549 cell line, Cytogeneticcharacteristics of A-549, MCF-7 and COLO-205 cell lines after theirexposure to the action of components containing ³⁹K, ⁶⁴Zn and ²⁴Mg,Example 4, Comparative characteristics of the action of Doxorubicin andthe action of components containing ³⁹K, ⁶⁴Zn and ²⁴Mg on normal ratkidney cells (NRK), Comparative assessment of the effects of ³⁹K, ⁶⁴Znand ²⁴Mg vs Doxorubicin, an anti-cancer agent, on normal rat kidneycells (NRK cell line), Example 5, Assessment of migrationcharacteristics of A-549, FC, NRK, HaCaT, A-431 and MM-4 cell linesafter their exposure to the action of components containing ³⁹K, ⁶⁴Znand ²⁴Mg. Analysis of the combined effect of each of the components of³⁹K, ⁶⁴Zn and ²⁴Mg and Doxorubicin, Assessment of the combined effect ofthe component containing ⁶⁴Zn and Doxorubicin, Assessment of the effectof ³⁹K, ⁶⁴Zn and ²⁴Mg on migration characteristics of stem cells derivedfrom rat fibroblasts, Combined effect of the component containing ⁶⁴Znand Doxorubicin on stem cells from rat fibroblasts, Migration activityof normal rat kidney cells (NRK cell line) after their exposure to theaction of components containing ³⁹K, ⁶⁴Zn and ²⁴Mg compared to theeffect of Doxorubicin, Combined effect of the component containing ⁶⁴Znand Doxorubicin on the migration activity of healthy human skin cells(HaCaT cell line), Combined effect of the component containing ⁶⁴Zn andDoxorubicin on the migration activity of human epidermoid carcinomacells (A-431 cell line), Combined effect of the component containing⁶⁴Zn and Doxorubicin on the migration activity of human melanoma tumorcells (MM4 cell line).

Referring now to Example 1, Burkitt lymphoma (Namalwa line) and acutepromyelocyte leukemia (HL-60 line) cells under the action of componentscontaining ³⁹K, ⁶⁴Zn and ²⁴Mg, the main objective was to assess thephenotypic differences between cells of Burkitt lymphoma and acutepromyelocyte leukemia under the action of components containing ³⁹K,⁶⁴Zn and ²⁴Mg.

The assessment of changes in the cell phenotype was carried out inrelation to the cytoskeleton and adhesion proteins in the human tumorcells of Burkitt lymphoma (Namalwa line) and acute promyelocyte leukemia(HL-60 line) after their exposure to the effect of components containing³⁹K, ⁶⁴Zn and ²⁴Mg.

The chemical form of the active ingredients is KCL for ³⁹K and sulfatesfor ⁶⁴Zn and ²⁴Mg. The following antigens/markers that distinguish adifferentiation status of the cells and their malignancy were used inthe research: ICAM-1 or CD54 marker is a single chain glycoprotein witha molecular mass of 55 kDa. It is an integral membrane protein thatcontains five extracellular Ig-like domains. ICAM-1 has a myeloid andB-cell origin on the tumor cells. In lymph proliferative diseases ICAM-1expression is associated with the degree of malignant transformation.ICAM-1 is expressed on various types of endothelial cells, epithelialcells, tissue macrophages, mitogen-stimulated T-cells, in the germinalcenter cells, dendritic cells and in lymph nodes. ICAM-1 fibroblasts andendothelial cells are induced by such inflammatory mediators as IL-1,TNFα and IFNγ: ICAM-1 expression increases during 6 to 8 hours afterstimulation and persists for at least 48 hours.

E- and N-cadherin family mediates cell-cell adhesion in the presence ofcalcium ions. The cadherin family consists of structurally similarmolecules composed of 723 to 748 amino-acid residues. The degree ofhomology between cadherins from a variety of tissues and samples reaches50 to 60%. The cadherin family includes three subclasses: E-cadherinsfound in epithelial cells and known as uvomorulin, a cell adhesionmolecule (CAM 120/80 or L-CAM), N-cadherins found in the mature nervousand muscular tissue and known as A-CAM and P-cadherins originally foundin the placenta and epithelium but expressed transiently by othertissues as well during their development. Cadherins play an importantrole in the formation of adhesive contacts responsible for theorganization of the cytoskeleton of cells. CD44 marker is a glycoproteinwith a molecular mass of 80 to 95 kDa and is a cell-surface marker forT-lymphocytes and B-lymphocytes, monocytes, macrophages, granulocytes,fibroblasts, epithelial cells and cells of the brain, red blood cells.

Alluding to the above, CD44 is involved in cell-matrix interaction,activation of lymphocytes and in lymph node homing. In addition toparticipating in the formation of physical contact between stromal cellsand early B-cell precursors, it is involved in other forms of cell-cellinteractions as well as in cell migration and metastasis processes. Inaddition, examination of these antigens from the perspective of researchinto the epithelial-mesenchymal transformation (EMT) process is ofimmediate interest since the above named proteins are also EMTassociated markers. It is important to note that EMT, a highly conservedcellular program that leads to the transformation of adherent polarizedepithelial cells into mobile morphologically altered mesenchymal stemcells, is mostly studied only in relation to solid tumors.

However, articles about an important role of EMT in the study oflymphomas and leukemias as well have started coming out recently, whichis an interesting stage in the research into the tumor process as awhole, since these tumors radically differ from “usual solid neoplasias”in their histogenesis and, accordingly, in their phenotypiccharacteristics. Thus, such comprehensive research into adhesionproteins and cell cytoskeleton in the tumor cells of Burkitt lymphomaand acute promyelocyte leukemia after the action of the group ofcomponents under study is a relevant and contemporary attempt of adeeper analysis of the modification of a cell phenotype of tumor cellsin order to assess changes in the degree of their malignancy. Materialsand methods for leukemia cell lines HL-60 and Namalva and cell cultureconditions will now be described. Initial HL-60 and Namalva cells werecultured in complete RPMI 1640 medium (PAA) (PAA, Austria) supplementedwith 10% fetal calf serum (PAA) and incubated in 5% CO₂ humidifiedatmosphere at 37° C. The culture medium was replaced in 2 to 3 days andthe cells were passaged in 4 to 5 days. The effect of ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents and doxorubicin (chosen as a reference drug) on viability ofcell lines HL-60 and Namalva was assessed using 2 plates (Type of cellplate-TPP, Italy). The first plate was for coloring cells with trypanblue and the second one was for the MTT assay.

To carry out this experiment, the components under study, namelychloride (for ³⁹K) and sulphates (for ⁶⁴Zn and ²⁴Mg), were introduced inthe respective wells of the 96-well plates for cell culture (evenly forthe 2 plates) according to the scheme shown below. Initialconcentrations of the components. Initial solutions of chloride (for³⁹K) and sulphates (for ⁶⁴Zn and ²⁴Mg) were administered at the doses of20 mg/ml, 4 mg/ml, 0.8 mg/ml, 0.16 mg/ml, 32 mcg/ml, 6.4 mcg/ml, 1.28mcg/ml, 0.256 mcg/ml to 100 mcl of complete medium (RPMI 1640 (PAA,Austria)+10% fetal calf serum (PAA, Austria)+40 mcg/ml of gentamicin(Pharmak, Ukraine).

These initial concentrations of the components were prepared separatelyin sterile microtubes before the start of the experiment. Initialconcentrations of doxorubicin. Initial (prior to the experiment)concentrations of doxorubicin prepared in a similar manner in sterilemicrotubes were as follows: 0.8 mg/ml, 0.16 mg/ml, 32 mcg/ml, 6.4mcg/ml, 1.28 mcg/ml, 0.256 mcg/ml, 51.2 ng/ml, 10 ng/ml and were alsoadded to 100 mcl of complete medium. Differences in initialconcentrations of the components under study and doxorubicin are due tothe various degree of their activity: we compared those concentrationswhich produced a statistically significant effect. The main purpose ofthe research was to assess the phenotypic changes in the cells afterthey were exposed to the action of the group of components under studycontaining ³⁹K, ⁶⁴Zn and ²⁴Mg. As is known (Nekludov A. D., 1990;Belokrylov G. A. et al., 1996; Kolganov A. S., 2001; Horwitz L. D.,1994; Zhou S. et al., 2001), doxorubicine has pronounced cytostaticeffect (starts the process of dying of both affected and healthy cellsas evidenced by the picture of their death in FIG. 10C, 10D, 10E, 10F)starting from the minimal doses of action of this component.

The cytotoxic properties of doxorubicin (especially its selectivecardiotoxicity and expressed depression of bone marrow function) werestudied in detail in several papers (Stukov A. V. et al., 1998; VatutinN. V. et al., 2001; Kolygin B. A., 2002; Lushnikova E. L. et al., 2004;Singal P. K., Iliskovic N., 1998; Lebrecht D. et al., 2004; Girl S. N.et al., 2004) and pose a major problem in its widespread use in cancerpatients due to reduction in the length and quality of life of thepatients and sometimes resulting in lethal outcome. Thus doxorubicin asa reference drug is characterized by a negative effect both on healthycells, as it triggers the mechanism of cell death, and on the wholebody.

After preparation of the corresponding concentrations of the components,they were introduced into appropriate wells of the plate in which HL-60or Namalva cell were in complete medium. The volume of the culturemedium with cells was 100 mcl per well and the concentration of cells inthe suspension was 3×105 cells/ml (3×106/plate). After the componentswere placed into the wells, the final concentration (component+cells inthe suspension) became smaller and corresponded to the values given inthe Table of Concentration Characteristics. For components containing³⁹K, ⁶⁴Zn and ²⁴Mg the values of final concentrations (chloride andsulfates) are shown in FIG. 1, in 1st, 2nd and 3rd column of the tableand for doxorubicin its concentrations in the wells.

After the components were placed into the wells, the plate was placed inan incubator in 5% COz humidified atmosphere at 37° C. Quantitativecharacteristics of live cells and dead cells (coloring of the cellsuspension with trypan blue) or their viability (MTT assay) Thequantitative assessment of live and dead cells after the action ofdoxorubicin was performed by their coloring with trypan blue (HyClon,USA), 20 mcl of the suspension of the test cells were mixed with 20 mclof the trypan blue solution and then resuspended. The obtained solutionwas placed into the Goryaev chamber and the number of live and deadcells was calculated using the following formula: A/80×2=X*106 cells per1 ml of medium, where A is a number of cells calculated in the Goryaevchamber (in 5 squares) and ×2 is trypan blue dilution (1:1). The cellviability after the action of the group of components under studycontaining ³⁹K, ⁶⁴Zn and ²⁴Mg was determined using MTT(Methylthiazoletetrazolium). To do this, 10 mcl of MTT solution (Sigma,USA) (5 mg/ml of dye in PBS) were added in each well of the plate. Thenthe plate was incubated in a CO₂ incubator at t 37° C. for 3 hours.

After the incubation the medium was removed from the wells and theformed tetraformazan crystals were dissolved in 50 mcl ofdimethylsulphoxide (Applichem, Germany). The results were fixed using amulti-well spectrophotometer with an excitation wave length of 540 nm.The percent of inhibition of cell viability was calculated using thefollowing formula: IR=(1−A 540 (experiment)/A540 (control))×100%. Incarrying out the immunocytochemical (ICC) assay, cells on microscopeglasses (cytospin preparations) were fixed in the solution(methanol+acetone (1:1)) for 2 hours at t−20° C. and then incubated witha 1% solution of bovine serum albumin (BSA) for 20 minutes.

Then such monoclonal antibodies as CD44, N-cadherin, ICAM and IgM wereapplied for the period of time specified in the manufacturer'sinstructions (30 to 60 minute time intervals), after which Poly Vueimaging system conjugated with peroxidase was used and the enzymeactivity was detected using diaminobenzidine (ThermoScientific) as asubstrate. After the immunocytochemical reaction, the preparations wererinsed with water and counterstained with hematoxylin and eosin for 15to 30 seconds. The results were analyzed by a quantitative estimation ofcells with the marker expression (increased brown color of cellscorresponded to a higher degree of expression of the marker) using alight microscope and assessed using the classical H-Score method:S=1×A+2×B+3×C, where S is the H-Score index the values of which arewithin the scope of 0 (protein is not detectable) to 300 (high-levelexpression in 100% of the cells), A is a percentage of weakly stainedcells, B is the percentage of moderately stained cells, and C thepercentage of strongly stained cells.

The result of comprehensive assessment of the effect of componentscontaining ³⁹K, ⁶⁴Zn and ²⁴Mg and doxorubicin, as a reference element,on the HL-60 and Namalwa tumor cells showed that the componentscontaining ³⁹K, ⁶⁴Zn and ²⁴Mg in most cases increased the amount ofN-cadherin-positive cells both in the Namalwa and in HL-60 cell lines.An exception was ³⁹K at a dose of 2 mg/ml and ²⁴Mg at the same dose of 2mg/ml in the Namalwa cell line. Increase in the number of cellsexpressing N-cadherin is first of all indicative of an effect of thesecomponents not only on the adhesion of cells and their cytoskeleton, butalso on the cell cycle programs, as N-cadherin has an inhibitory effecton cell proliferation in mesenchymal (malignant) cells, i.e., itsoverexpression suppresses cell proliferation by extension of G2/M phasethrough activation of catenin-dependent expression of p21 (an inhibitorof cyclin-dependent kinase).

Thus N-cadherin-dependent signaling pathways inhibit tumor cellproliferation due to deceleration of G2/M phase and prevent tumor growthby stopping cells in their specific points of the cell cycle. As forICAM (another adhesion marker), an interesting data was obtained on thereduction in the number of cells that express this marker after they areexposed to the action of components containing ³⁹K (at the two doses—2mg/ml and 0.5 mg/ml) and ⁶⁴Zn (at a dose of 10 mcg/ml) which isindicative of a dose-related effect in suppression of the malignantphenotype of HL-60 cells.

Values of expression at the control level are shown in the HL-60 cellline by the component containing ²⁴Mg. All the tested components in theBurkitt lymphoma tumor line (Namalwa) showed decrease in the expressionlevel compared to the control group of cells. Reduced compared with thecontrol values, parameters of expression of the ICAM adhesion marker arecharacterized as a positive point in terms of a prognostic factor ofdevelopment of atherosclerotic vascular changes in cardiac and vascularpathologies (coronary heart disease, thrombosis, venous varices,coronary syndromes) and reflect the decline in metastasis processes andmalignant potential of cells. Increase in the number of ICAM-positivecells with the action of component containing ⁶⁴Zn as sulfate at a doseof 5 mcg/ml on the Namalwa cell line (up to 210 according to the H-Scoremethod) may be indicative of an enhancement of reactivity of the immunesystem and the targets for the anticancer targeted therapy. The resultof effect of ICAM marker on HL-60 was a decrease in the number of cellsexpressing it for the entire test group.

The result of effect of components containing ²⁴Mg and ⁶⁴Zn as sulfateswas a significant change in the morphological and growth characteristicsof the cells: there was a significant enhancement of adhesion of cellsto the substrate and their spreading all over the culture plasticsurface, as shown in Fures 101, 10J, 10K, 10L, which was observed on theliving cell culture.

The adhesive characteristics of tumor cells directly correlate withtheir invasion, migration and, as a consequence, with metastasis andrecurrence in the development of neoplastic process. The process ofepithelial-mesenchymal transition (EMF) of tumor cells, which alsoinvolves cell adhesion proteins and cytoskeleton, plays a key role inthe metastatic cascade. During changes in the cell morphology, (both:when adhering to a substrate and when spreading on the surface) themembrane properties and the cell cytoskeleton also change, which in itsturn results in changes in the receptor profile of cells, andvariability of various signaling pathways of cells including thosepathways that are associated with an increase in the sensitivity ofcells to antitumor agents.

Strengthening of the adhesive properties of cells both among themselvesand to the culture plastic, characterizes the decrease in their tumorand metastatic potential, and is also indicative of an increase in thedegree of differentiation of cells and thus of a decrease in theirmalignant phenotype, and the fact of change in the cell sensitivity toanticancer preparations when their morphology changes is still of greatinterest. Morphological and phenotypic changes in the cells after theaction of components containing ²⁴Mg and ⁶⁴Zn provide opportunities forcombined use of the components under study and officially registeredchemotherapeutic agents as a complex therapy which consists insequential administration of the component under study (²⁴Mg or ⁶⁴Zn)followed by a well-known anticancer agent. The proposed complex approachis based on the increased sensitivity of tumor cells to an antitumortoxic agent at the first stage due to the use of ²⁴Mg or ⁶⁴Zn and aneffect of the official antitumor agent at the second stage.

Considering fact of negative effects of any official antitumorpreparation on the human body, by increasing the sensitivity of cells,e.g., to doxorubicin, via ²⁴Mg and ⁶⁴Zn at the first stage we reduce theamount of chemotherapeutic agent injected into the patient's body at thesecond stage of the treatment.

Thus, the result of the proposed complex approach will be a decrease inthe toxic effects of known anticancer agents on the human body. Thecombined (alternate) use of components containing ²⁴Mg and ⁶⁴Zn looksquite promising in terms of the synergistic effect. By significantlychanging the cell morphology via component ²⁴Mg at the first stage (inthe absence of its toxic properties) and using a combination with thehighly effective ⁶⁴Zn in the second stage (so far as ⁶⁴Zn is close todoxorubicin in an effective dose of exposure but without a negativeeffect on healthy cells of the body) one can obtain a highly effectivecomplex of ²⁴Mg+⁶⁴Zn with a potential modifying effect on the process ofepithelial-mesenchymal transition with antitumor and antimetastaticproperties.

Also interesting was the fact of significant inhibition of expression ofCD44 marker in Namalwa cells after they were exposed to the action ofcomponents containing ³⁹K and ⁶⁴Zn and a tendency to decrease in thelevel of expression of the protein in the cells of HL-60 line(especially in high doses). Since this antigen in the tumor cells isoften associated with an aggressive, malignant phenotype, thus itsdecrease is a favorable factor in predicting the flow of a tumor processas a whole. Characteristics of IgM marker in the Namalwa cell lineshowed a decrease in its expression compared to the control group ofcells. Inhibition of IgM is associated with induction of celldifferentiation as well as activation of the signaling pathways ofapoptosis.

Thus through the example of all the components under study we showed theactivation of apoptotic pathways that brings a tumor cell closer to thestate of its natural programmed death by reducing its malignant andmetastatic potential. Phenotypic Features of Human Burkitt LymphomaCells (Namalwa Line) under the Action of Components Containing ³⁹K, ⁶⁴Znand ²⁴Mg Results of Immunocytochemical Analysis of Adhesion Proteins andCytoskeleton of N-cadherin and ICAM in Namalwa Cells after the Action ofComponents Containing ³⁹K, ⁶⁴Zn and ²⁴Mg.

FIG. 10 illustrates a morphological and growth characteristics of cellsof HL-60 cell line after their treatment with components containing ³⁹K,⁶⁴Zn and ²⁴Mg A: Control cells of original HL-60 cell line (×10), B:control—cells of original HL-60 cell line (×40), C: HL-60cells+doxorubicin at a dose of 0.08 mg/ml (×10), D: HL-60cells+doxorubicin at a dose of 0.08 mg/ml (×40), E: HL-60cells+doxorubicin at a dose of 32 mg/ml (×10), F: HL-60cells+doxorubicin at a dose of 3.2 mg/ml (×40), G: HL-60 cells+componentcontaining ³⁹K as chloride at a dose of 2 mg/ml (×10), H: HL-60cells+component containing ³⁹K as chloride at a dose of 2 mg/ml (×40),I: HL-60 cells+component containing ⁶⁴Zn as sulphate at a dose of 16mcg/ml (×10), J: HL-60 cells+component containing ⁶⁴Zn as sulphate at adose of 16 mcg/ml (×40), K: HL-60 cells+components containing ²⁴Mg assulphate at a dose of 2 mg/ml (×10), L: HL-60 cells+componentscontaining ²⁴Mg as sulphate at a dose of 2 mg/ml (×40).

Following the results of immunocytochemical analysis of adhesionproteins and cytoskeleton obtained experimentally as a result of actionof the group of components under study containing ³⁹K, ⁶⁴Zn and ²⁴Mg onthe cell lines of human Burkitt lymphoma (Namalwa line) and acutepromyelocyte leukemia (HL-60 line) we can state the main findings: Viaexample of N-cadherin marker, we demonstrated an increase in theadhesion properties of Namalwa and HL-60 tumor cells both amongthemselves and to the culture plastic, which directly correlates withthe reduction of invasive and metastatic properties of tumor cells.

A decrease in ICAM marker expression (compared to the control cells)after the use of components containing ³⁹K, ⁶⁴Zn and ²⁴Mg is a positiveresult in terms of the following forecast: the risk of atheroscleroticchanges in the cardiovascular system in the form of thrombosis, varicoseveins and coronary syndromes sufficiently reduced; an increase in themarker expression on most cell lines is associated with an increase intheir malignant potential, therefore, the result of reduction of suchparameters after the action of the group of components under study isindicative of the attenuation of malignant properties of tumor cells.

Due to changes in the sensitivity of cells to antitumor agents under theeffect of ⁶⁴Zn and ²⁴Mg (for example, changes in the cell morphology)there is a possibility of the combined therapy that involves theintegrated use of the group of components under study in combinationwith the official antitumor drugs, which will result in reducing thenegative impact of antitumor components by down-titrating the latter.There are changes in the cell morphology (in the form of enhancement oftheir adhesiveness both among themselves and to the culture plastic)after the action of ⁶⁴Zn and ²⁴Mg which is indicative of a decrease inthe metastatic potential of tumor cells.

A decrease in the level of expression of CD 44 marker after the actionof components containing ³⁹K, ⁶⁴Zn and ²⁴Mg, which in the tumor cells isassociated with a more aggressive and malignant phenotype, characterizessaid light isotopes as inhibitors of malignant and invasive properties.Via example of IgM marker, we showed an activation of signaling pathwaysof apoptosis after the use of components containing ³⁹K, ⁶⁴Zn and ²⁴Mgin the Namalwa tumor cell line which is confirmed by a decrease in theexpression level of the marker. Activation of the apoptotic pathwaycontributes to the return of the cell to a state of its naturalprogrammed death thus reducing its tumor and metastatic potential.

Assessment of antitumor and biological activity of components,containing ⁶⁴Zn, ²⁴Mg and ³⁹K and Zn, Mg, and K elements on a model ofrenal cell carcinoma (PA) in a rat in an in vitro experiment will bediscussed. The data represented in this section allows to o assess thedegree of antitumor and biological activity of ³⁹K, ⁶⁴Zn and ²⁴Mgcomponents from the standpoint of their changing the morphology andphenotype of tumor cells of PA renal cell carcinoma in the in vitroexperiment and compare the obtained result with the effect of K, Zn andMg elements which have a natural distribution of isotopes. It alsoallows making a quantitative assessment of changes in the cellsphenotype in terms of correlation between the number of the actingcomponents and the number of transformed cells.

By using different methods of staining cells after their exposure to theaction of the components, to make a comparative assessment of cellsafter treatment. To compare the obtained result with the effects of theofficial antitumor agent doxorubicin EBEWE. In this example, we hadpotassium, magnesium and zinc in 2 groups: Group No 1 contained lightisotope components ³⁹K, ⁶⁴Zn and ²⁴Mg, and Group No 2 contained the sameelements-K, Zn and Mg but with a natural distribution of isotopes.Doxorubicin Ebewe (Austria), an official anticancer chemotherapeuticdrug, was used for reference. Light isotope components as well as zincand magnesium, components with the natural distribution of isotopes,were used in the form of sulphates, and potassium as a chloride. 0.9%NaCl solution supplemented with 5% glucose was used as a solvent.

Staining of Cell Suspension with Trypan Blue and Visual Control ofChanges in the Shape, Color and Size of the Cells will now be discussed.The visual control method was based on the observation of state of theplasma membrane permeability after the action of materials under study,and reflects the degree of membrane's damage, making it possible toestimate an ability of nuclear proteins to absorb the dye.

To make the analysis, the cell suspension, after it was exposed to theaction of materials, in a volume of 20 mcl was mixed with 20 mcl oftrypan blue solution and resuspended. The obtained solution was placedin a hemocytometer chamber for visual analysis and quantitativeestimation of the cells. To make the quantitative estimation of thecells, they were separated from the substrate using Versene solution andcounted by staining cells with trypan. A change in the color of a cellwas indicative of the violation of intactness of the cell membrane, andthe more pronounced was the coloration, the less viable the cell was.Undamaged parenchymatous cells had a convex, well-defined surface linethat reflected light and colored ranging from yellow to nearlytransparent color. They are easily distinguishable from dark damagedcells and easily identified with ordinary light microscope. This methoddoes not characterize any changes in the cell phenotype.

Alluding to the above, staining the Cells with Crystal Violet and theirQuantitative Analysis will be discussed. Utilization of this method ofassessment in the experiment makes it possible to quantify cellsclassified according to the living-dead criterion. The method is basedon statistical evaluation of cells able to adhere to the substrate (tothe plastic plate), cells capable of further proliferation. Thecriterion of the evaluation is the degree of optical permeability of awell with the cell sediment for a luminous flux of a certain wavelength.Removal of the culture medium from the wells is also accompanied byremoval of non-adhering and unstained cells from the substrate, andadhesion and staining of the residue left on the substrate provides aquantitative characteristic of the number of cells which are capable offurther division and multiplication.

Alluding to the above, viable tumor cells capable of furtherproliferation were fixed to the plastic surface and stained blue (afterthey were exposed to the action of components with natural distributionof isotopes) using the dye powder dissolved in 70% methanol solution.After 10 minutes the dye was removed from the wells with a micropipetteand the cells were thoroughly rinsed three times with clean runningwater. The cell sediment fixed by the dye was dissolved with ethanolfollowed by determination of its concentration (which is indicative ofthe number of viable cells) and fixed via multi-well spectrophotometerwith an excitation wave 540 nm in length.

Since the method does not characterize an appearance and shape of thecells after their treatment with the medication under study, as shown inFIG. 11, the assessment was carried out only for the components with thenatural distribution of isotopes without consideration of light isotopecomponents.

Primary cell culture for our in vitro experiments was obtained byenzymatic treatment of tumor material (using trypsin solution) from astrain of renal cell carcinoma (PA) in a rat Visually (after its removalfrom the animal), the tumor looked as a nodular fleshy growth of awhitish-pink color of rather dense consistency, marginated from othertissues by a joint capsule. The main morphological signs of PA tumor inthe animal can be described as follows. Small (1.5 to 2 grams) fragmentswithout vessels and blastic lesions were excised aseptically from thesurgically removed tumor and then cut them with scissors to smallerpieces of 0.2 to 0.3 mm in size and cultured in a solution of trypsin.The culture medium contained the following components: RPMI-1640 (Sigma,USA) as the base plus 10% newborn calf serum and 40 mcg/ml ofgentamicin. Trypsinization was performed in a humidified atmosphere of5% CO₂ at 37° C. with constant mixing of the contents in a magneticstirrer. The isolated cells were washed three times in a culture mediumby centrifuging and before starting the experiment a quantitativeassessment of cells was carried out in the Goryaev chamber withsupravital staining of the cells with trypan blue.

Complex clusters of polymorphic cells dispersed in the form of separateislands separated from each other by layers of edematous connectivetissue were detected in the main part of the tumor. Most of the tumorfeatured pronounced polymorphism which was expressed as significantdifferences both in cell shapes and sizes. We detected polygonal, ovaland round cells, as well as their components which can be attributed toatypical fibroblasts. The cell nuclei had a considerable variationdepending on the size of the cell itself, and most of them were largeenough and occupied the main volume of the cell. Chromatin of the cellshad a grid structure, the cytoplasm was characterized by a pronouncedbasophile.

Stroma and tumor borders had quite an extensive network of blood vesselslike capillaries through which the tumor tissue was fed. The edematousconnective tissue contained a lymphoid net. A granulation connectivetissue containing lymphoid cells was detected along the perimeter. Therewere some figures of cell division as shown in FIG. 12. A tumor growthchart built on the results of observations of an animal with thetransplanted tumor for 29 days clearly describes the kinetics of thetumor growth as shown in FIGS. 13, 14.

The suspension of tumor cells was transplanted into the animal weighing120 g, and according to the data obtained during the 29 day it increasedin volume to almost 120,000 mm3 which corresponds to the tumor weight of120 g. The obtained result characterizes a fairly high degree of itsmalignancy with its growth under in vivo conditions as such dynamics ofgrowth is not peculiar to most transplanted strains. We also made aquantitative estimation of the ability of tumor cells to furtherproliferation. Transplantation of a tumor strain into the animal wascarried out using the cells frozen at −196° C. After thawing of theampoule it was noted that the cell suspension had a sufficiently lownumber of tumor cells capable of further division which was confirmed bycalculation of their number in the Goryaev chamber.

Analysis of the thawed suspension was conducted by staining the cellswith trypan blue and it showed that after thawing a number of viablecells in the suspension did not exceed 150 thousand, which isapproximately 10% of the total number of cells. Development of the tumorprocess, the beginning of which was observed as early as on the 5th dayafter 150 thousand unfrozen cells were transplanted into the animal,also characterizes a fairly high degree of malignancy of the tumor. Thecell material used in further in vitro experiments was obtained afterthe third passage of the strain in vivo.

Analysis of the effect of all components on the renal cell carcinoma(PA) was conducted to determine the relationship between the amount ofthe preparation (concentration characteristics of the component) and thequantitative characteristics of the cells. Assessment of the effect ofthe components was carried out on 2 concentration models with 30 and 300thousand original tumor cells. After the cells were cultured, cellsuspensions were prepared in the plates with the culture medium and aneffect of the preparations (in various concentrations) on the tumorcells was assessed. The experiment was started with the concentration of10 mg of the active substance per 1 ml of the solvent (saline with 5%glucose).

The amount of culture medium in each well was 200 mcl for 30 thousandcells and 2 ml for 300 thousand cells, respectively. The amounts ofdrugs (active substances) for 2 concentration models differed accordingto the amount of cells and culture medium, i.e., the amount of drug inthe experiment with 300 thousand cells was ten times as large ascompared with the same in the experiment with 30 thousand cells. Theresult of effects of the components is shown in FIGS. 22, 23 for lightisotope components and in FIG. 25 for the elements with naturaldistribution of isotopes. Assessment of the effect of the components wasperformed by comparing the external factors of cells (color, size,shape) prior to the experiment and after staining.

As a result of the action of ⁶⁴Zn, ²⁴Mg and ³⁹K on the PA tumor cells,after their staining with trypan blue, the presence of cells of 2 typesreferenced by letters “A” and “B” as shown in FIGS. 16, 17, 18, wasdetected. Classification of the cells after their exposure to Zn, Mg andK elements with the natural distribution of isotopes and DoxorubicinEbewe was carried out by the living-dead criterion. These differencesare shown in FIGS. 19, 20. The initial concentration of the componentsin the experiment was 10 mg of the active substance (sulphate orchloride) per 1 ml of saline and it was changed towards decrease inaccordance with the “component dose” column in FIGS. 22, 23, 25. Changesin the appearance of cells from the initial tumor cells to the cellswith morphology of types A and B were noted with regard to all lightisotope components but with the following differences.

To obtain an accurate statistics, the experiment was conducted in 2concentration models: for 30 thousand and 300 thousand cells. ²⁴Mg and³⁹K components in both concentration models, after their adding into thewells with the cell suspension and first calculation conducted 10 hoursafter the start of the experiment, showed 100% concentration of thecells of type A. Effect of ⁶⁴Zn (also shown in both concentrationmodels) consisted in its ability to transform cells from the initialtumor cells into cells of type A. Cells of type B were detected withinthe concentration range of 10 mg/ml to 0.4 mg/ml. The concentrationrange of type A cells was from 0.08 mg/ml to 0.025 mcg/ml. Registrationand calculation of the quantitative characteristics of were carried outevery 10 hours, and the total time of the experiment was 140 hours witha total of 14 plates. All nine concentrations were used within one96-well plate which is reflected in tables and graphs. A quantitativeassessment of cells of each type which was conducted with 10-hourintervals. The concentrations of the substances were tested in thefollowing order: 10 mg/ml, 2 mg/ml, 0.4 mg/ml, 0.08 mg/ml, 16 mcg/ml,3.2 mcg/ml, 0.64 mcg/ml, 0.128 mcg/ml, 0.025 mcg/ml.

Referring now to FIGS. 22, 23, 24 the presentations shown there on madeto show the concentration effect of ⁶⁴Zn, ²⁴Mg and ³⁹K components on the2 models (for 30 000 and 300 000 cells in the culture medium). Analysisof the malignancy of cells of type B was planned to be made in a testfor tumorigenicity in the agar medium. Since the PA cell line showed theinitial development of colonies in the agar medium starting with aconcentration of 500 thousand cells and their weak growth even in thisconcentration, it was decided to make an assessment of malignancy basedon the level of expression of markers in 3 human cell lines.

After the action of components with natural isotope distribution on PAtumor cells (in all 9 concentration dilutions of the preparations), theywere stained with trypan blue. Staining cells (after the effect of thecomponents) in blue is indicative of the respiratory impairment of thecells, which allows of classifying them as non-viable tumor units. Thepart of the cells remained uncolored is characterized as viable with theability for further proliferation. The results presented in FIG. 25 andin FIG. 26 show interrelation between the active dose of the componentswith natural isotope distribution and a number of viable tumor cells.The assessment was performed on 14 plates with 300 thousand cells ineach well by counting the number of viable cells in the Goryaev chamber.Quantitative analysis of the cells was performed visually every 10 hoursof the experiment. Statistical quantification of both viable and unableto reproduction tumor cells was performed using 2 methods: staining thecells with trypan blue and crystal violet. The photomicrographs of cellsin the Goryaev chamber made after staining clearly characterize aclassical picture of violations of the integrity of the membrane, whichgives reason to characterize cells colored in blue as non-viable, asshown in FIG. 19, 20.

In order to make a comparative (concentration and visual) assessment ofcells, the action of antitumor drug Doxorubicin Ebewe was tested. Theconcentrations of Zn, Mg and K elements (with natural distribution ofisotopes) and Doxorubicin Ebewe showed the following. The initialconcentration of substances was 10 mg per 1 ml of solvent. According tothe data in FIGS. 25, 28, which generalize the statistical estimation ofthe number of live tumor cells performed by staining cells with crystalviolet and trypan blue, Doxorubicin Ebewe kills 50% of the tumor cells(leaving unaffected the remaining 50% viable tumor cells) at theconcentration of 3.2 mcg/ml. Elements Zn, Mg and K with the naturaldistribution of isotopes demonstrated much lower ability to kill tumorcells, which was exhibited in their significantly higher concentrationsas shown in FIG. 28. The main differences of the effects of ⁶⁴Zn, ²⁴Mgand ³⁹K on the PA tumor cells consisted in the following. The ability ofthe initial tumor cells to transform into the cells with the changedcolor was noted. It was also found that the cell adhesion to the cultureplastic had a different character as well.

The effects of ⁶⁴Zn, ²⁴Mg and ³⁹K in two concentration models are shownin FIGS. 22, 23 that shows the results of the experiment. The changedcharacter of the cell adhesion after treating the cells with all thelight isotope elements is shown in FIG. 36 of the presentation and ischaracterized by the presence of the cells spread on the cultureplastic. In order to better display the whole picture of concentrationin the experiment, the main results of effects of the components areshown in 2 tables: see FIG. 27, 28 on ⁶⁴Zn, ²⁴Mg and ³⁹K and Table 3.2on K, Zn and Mg (with the natural distribution of isotopes).

The number of cells after the action of the preparations was calculatedat each control point (covering all 96 wells of the plate and all 9concentrations of each component). The number of type A cells wasdetermined after the action of ³⁹K, ⁶⁴Zn and ²⁴Mg and the number ofviable cells—after the action of K, Zn and Mg with a natural isotopedistribution. Characteristics of changes of the cell phenotype throughtime after they were exposed to the action of ³⁹K, ⁶⁴Zn and ²⁴Mg are asfollows. The main result, which consists in the transformation of 100%initial tumor cells in the type A cells was detected with the lightisotope group of components within the first 80 hours of the experimentwith the following differences for each of the light isotopes as shownin FIG. 30.

Cells exposed to the action of ⁶⁴Zn showed 100% change in theirphenotype during the first 50 hours of the experiment with 80% of saidchanges during first 10 hours. Transformation time of the initial tumorcells into cells of phenotype A after the action of ³⁹K was 80 hours,and after the action of ²⁴Mg-70 hours. Beside the time interval duringwhich transformation from the initial tumor cells into cells ofphenotype A was observed, the stability of the obtained results waschecked. Thus out of the total number of control points which was equalto 14, the number of additional monitoring points for zinc was 9 forpotassium-6 and for magnesium-7.

According to monitoring of the results during the first 80 hours therewere cell changes from the original tumor cells to cells of type A andno further changes were observed.

Quantitative analysis of the cells at the last 14th point of theexperiment confirmed that the cells maintained the acquired phenotype.No quantitative changes in the opposite direction were detected.

The data about kinetics of the cell transformation process caused by³⁹K, ⁶⁴Zn and ²⁴Mg components are illustrated in FIG. 30. The experimenton assessment of the effects of K, Zn and Mg elements (with the naturaldistribution of isotopes) on renal carcinoma cells (PA) was conducted ina similar manner but with a quantitative assessment of viable tumorcells. The assessment criterion in this case was the integrity of thecell membrane which did not allow the dye to penetrate deep into cellsand stain them. Dependence of the number of viable tumor cells versusthe time of action of the components is shown in FIG. 29.

The results of effects of K, Zn and Mg with a natural distribution ofisotopes and the reference drug Doxorubicin Ebewe on a strain of renalcell carcinoma (PA) manifested themselves as follows. Dynamics of therate of activity of K, Zn and Mg elements with a natural distribution ofisotopes did not differ significantly within the group of 3 elements.All components showed their ability to kill 100% of tumor cells within atime range of 105 to 112 hours.

According to FIG. 25 and summarized data in FIG. 28 the reference drug,Doxorubicin EBEWE, showed the highest activity, which at a dose of 3.2mg/ml killed 50% of tumor cells. The doses of other elements weresignificantly higher to achieve the same effect. Zn showed the sameability (50% live, 50% dead) at a dose 100 times as high (300microgram), the dose for Mg will be 1600 times as high (about 5 mg), andfor K-650 times as high (about 2 mg). Doses required for complete 100%elimination of tumor cells are roughly the same.

A comparative assessment of the ability of light isotope components tochange the phenotype of initial PA tumor cells to cells of type A wasmade based on data of concentrations of active substances andquantitative characteristics of the cells after their exposure to theaction of the materials. The result is shown in FIG. 30.

DNA Analysis of Stem and Experimental Cells using Fluorescent DyeHoechst 33258 will now be discussed. The main objectives was to studythe quality (visual) picture of the structure of DNA (genome) in avariety of cellular structures due to the formation of a stable complexbetween DNA and the fluorescent nucleotide-specific dye. The dye,entering the cell through the membrane, is selectively embedded alongthe minor groove of the double helix of DNA and thus makes it possibleto use the method for staining both nucleus and the surrounding cellareas. The molecules of the dye tied with DNA thus exhibit sufficientlystrong fluorescence when exposed to UV light. Characteristics of theintensity of the fluorescent staining allow for qualitative comparativeassessment of the genomes belonging to different types of cells. Theadvantages of the method are its rapidity and sensitivity to smallamounts of DNA in the test items. Comparative assessment of the genomewas determined on the following cell types: human stem cells, cells ofrenal carcinoma (PA) cell line after their exposure to the action of³⁹K, ⁶⁴Zn and ²⁴Mg, cells of renal carcinoma (PA) cell line after theirexposure to the action of K, Zn and Mg with a natural distribution ofisotopes, and cells of renal carcinoma (PA) cell line after theirexposure to the action of anticancer drug Doxorubicin EbEWE.

The sequence of actions when staining the preparations consisted of thefollowing steps.

Cells, after their incubation with the components under study, werewashed from the culture medium in PBS by centrifugation at 1000 rev/minwithin for 7 minutes. Then a working solution of Hoechst 33258 in avolume of 200 mcl was added to the cell sediment and incubated in CO₂incubator for 30 minutes at 37° C. After incubation, the cells werewashed with PBS. Prior to photographing of the cells, their sediment wasdiluted in 30 mcl of glycerol and applied onto a specimen slide. Thepreparation was covered with a cover glass with glycerin to prevent airbubbles. The microscopic examination of the preparations was carried outusing fluorescence microscope Axiostar plus with a UV lamp. Thecomparative analysis made using Hoechst 33258 dye revealed thefollowing.

Assessment of the color intensity of human stem cells and tumor cells ofrenal carcinoma (PA) after their exposure to the action of ³⁹K, ⁶⁴Zn and²⁴Mg shows similarity in the intensity of fluorescence. Utilization ofthe reference drug Doxorubicin Ebewe, as shown in FIGS. 15, 20, 31, 32show induction of apoptosis and the presence of apoptotic bodies.Assessment of action of the dye was conducted on the preparations withthe active substance concentration that caused 50% cell transformationto phenotype A for the light isotope components and at a dose of 50 ICfor the elements with a natural distribution of components.

Comparative analysis of the appearance, color and shape of cells underthe action of ⁶⁴Zn, ²⁴Mg and ³⁹K, after cells were stained with crystalviolet demonstrated that the effect of said components consist in theformation of cells of 2 types which in their appearance differ from thecells exposed to the action of Zn, Mg and K elements with a naturalisotope distribution.

The action of ⁶⁴Zn produced largest morphological differences betweencells prior and after the exposure. By visual observation of the cellsafter the action of ⁶⁴Zn, ²⁴Mg and ³⁹K they were divided into groups Aand B. The effect ofZn, Mg and K elements with a natural isotopedistribution on PA tumor cells (stained with trypan blue) consisted inthe damage of the cells membrane. The cell classification was performedaccording to the live-dead criterion. Action of materials containing6Zn, ²⁴Mg and ³⁹K on the cells of a tumor strain of renal cell cancenoma(PA) produced an increase in the degree of their adhesion as comparedwith the action of Zn, Mg and K elements with a natural distribution ofisotopes.

Morphological differences after the exposure of cells to the action of⁶⁴Zn, ²⁴Mg and ³⁹K, as compared with Zn, Mg and K elements with anatural distribution of isotopes, were expressed by the increased degreeof adhesion of cells to the culture plastic surface. In most cases atumor growth in a biological organism is associated with the transportof cells via the blood circulatory and lymphatic systems and formationof secondary lesions (metastases). Materials containing light isotopesare effective in strengthening structural integrity of the tissue and insuppression of tumor cells migration through the bloodstream, i.e. inthe prevention of metastasis. ⁶⁴Zn containing material is capable ofmodifying the phenotype of original tumor cells. In vitro experimentdemonstrated ability of this component to transform initial tumor cellsof renal cell carcinoma (PA) in a rat into cells of 2 phenotypes. It isefficient within the same concentration range as Doxorubicin EBEWE.

Comparable concentration ranges in which the antitumor effect ofDoxorubicin EBEWE and the components of the test group were detectedwere as follows: the concentration range in which the effect ofDoxorubicin EBEWE was detected was from 16 mcg/ml (5% live and 95% deadcells were detected) to 0.64 mcg/ml (61% live and 39% dead cells weredetected), which is shown in FIG. 28. Using Doxorubicin inconcentrations below 0.64 mcg/ml showed that all tumor cells remainedviable. All tumor cells beyond the upper limit of the range of action ofDoxorubicin (concentration above 16 mcg/ml) were dead.

Zn with a natural distribution of isotopes (in sulfate form) showed aneffect similar to that of Doxorubicin which consisted in the presence oflive and dead cells after the action of sulfate, and the differenceswere recorded within the range. The upper point at which the start ofthe action of zinc was detected had the concentration of 80 mcg/ml andwas characterized by 25% of live tumor cells and 75% of dead tumor cells(based on trypan blue staining). The lower limit of the concentrationrange was expressed by the value of 16 mcg/ml with which 7% of deadtumor cells and 93% live tumor cells were detected. Beyond the lowerlimit of the range (less than 16 mcg/ml) all tumor cells were viable andbeyond the upper limit (80 mcg/ml) all cells were dead. DoxorubicinEbewe produces a similar effect outside the upper and lower limits.

Action of ⁶⁴Zn in the concentration of 3.2 mcg/ml resulted in a changein the cell morphology and phenotype. The cells transformed into 2 typesof cells the first of which (type A), based on the results ofcomparative analysis of the color and shape, has signs of similarity tothe stem cells. ²⁴Mg and ³⁹K components possess similar properties butwhen taken in much higher concentrations. Thus, the action of ⁶⁴Zn ismost effective in terms of specific characteristics of a mass unit ofthe substance able to transform the initial tumor cells into A cells.

The kinetic curves of the rate of transformation of initial tumor cellsinto cells with an altered phenotype (after the action of ⁶⁴Zn, ²⁴Mg and³⁹K) showed that for ⁶⁴Zn 80% of changes occurred in the first 10 hoursof the experiment. The rate indicators of cell transformation for ²⁴Mgand ³⁹K were 62% and 40% respectively over the same time. Analysis basedon staining cells with Hoechst 33258 dye revealed a similarity in theintensity of fluorescence of human stem cells and cancer cells exposedto the action of ⁶⁴Zn, ²⁴Mg and K.

The fluorescent ability of cells after their treatment with thecomponents with a natural distribution of isotopes was significantlylower. Dynamics of the activity of ⁶⁴Zn, ²⁴Mg and ³⁹K components withregard to their ability to transform initial PA tumor cells into cellsof phenotype A showed 80% of the transformation in the first 10 hoursfor ⁶⁴Zn, in the first 20 hours for ³⁹K and in the first 45 hours for²⁴Mg out of the total time of the experiment which equalled 140 hours.

The highest efficiency among ⁶⁴Zn, ²⁴Mg and ³⁹K (with regard to theability of the components to transform initial tumor cells into those ofan altered phenotype) was observed for ⁶⁴Zn. Assuming 50% of the cellstransformed from tumor cells to A cells, 1 microgram of ⁶⁴Zn sulfatedemonstrated a possibility of obtaining 23438 cells of type A, andassuming 100% transformation with the same amount of ⁶⁴Zn sulfate, only938 cells of type A can be obtained or 25 times less. It means there isnon-linear dependence between dosage of isotope containing material andefficiency of treatment.

Comparative assessment of the genome, carried out based on the intensityof fluorescence using Hoechst 33258 dye demonstrates similarity of thephenotype of stem cells and cells of phenotype A produced in result ofthe action of ⁶⁴Zn, ²⁴Mg and ³⁹K on cancer cells. The dynamics of growthof the PA tumor strain in the in vivo experiment showed the following:development of the tumor process in an animal with a transplantedunfrozen strain was initiated with 10% of viable cells; biochemicalkinetics of the PA tumor growth for 29 days showed an increase in itsweight to 120 grams, and initial signs of the tumor growth after thetransplantation (start of increase in weight) were observed already onthe 5th day. All these parameters in combination confirm an extremelyhigh degree of malignancy of the strain of renal cell carcinoma (PA).

Example 2, Section 2 will now be discussed. Immunocytochemistry ofadhesion proteins, cytoskeleton (cadherins) and CD 44 stem cell markerafter their exposure to the action of ³⁹K, ⁶⁴Zn and ²⁴Mg on a model ofrenal cell carcinoma (PA) in a rat. Detection of cumulative propertiesof ⁶⁴Zn. Cadherins are a group of glycosylated proteins and they aremainly responsible for the formation of cell-cell interactions providingadhesion between two identical molecules that are expressed on thesurfaces of two cells of one and the same type. Besides the physicalcontact that cadherins provide between the two cells, they facilitatethe transfer of a number of signals through cytoskeletal structures thuscontrolling cell growth and differentiation. Cadherins appear mainly inintercellular adhesion at the stages of morpho- and organogenesis. Theyprovide structural integrity of tissues (especially the epithelialmonolayer).

When making the immunocytochemistry (ICC) assay, cells on microscopeglasses (cytospin preparations) were fixed in the solution(methanol+acetone 1:1) for 2 hours at t 20° C. and incubated with 1%solution of bovine serum albumin (BSA) for 20 minutes. Then themonoclonal antibodies—CD44 (Diagnostic BioSystems), E-cadherin(ThermoScientific), N-cadherin (NeoMarker, BioLegend)—were applied for60 minutes, after which the Poly Vue imaging system conjugated withperoxidase was used and the enzyme activity was detected usingdiaminobenzidine (ThermoScientific) as a substrate.

After the immunocytochemical reaction the preparations were washed withwater and counterstained with hematoxylin-eosin (for 15-30 seconds). Theresults were analyzed by counting cells with expression (brown coloredcells), using a light microscope and were assessed using the classicalH-Score method: S=1×A+2×B+3×C, where S is the H-Score index giving arange of 0 (protein is not detected) to 300 (high-level expression in100% of the cells); A is the percentage of weakly stained cells, B isthe percentage of moderately stained cells and C is the percentage ofstrongly stained cells.

Three components-⁶⁴Zn, ²⁴Mg and ³⁹K at doses D1 and D2 were used, andthe action of ⁶⁴Zn was tested on 2 models: on freshly prepared solution(which was tested immediately after it was prepared) and on a solutionwhich was stored for 14 days at temperature+4° C. ⁶⁴Zn solution after 14days of storage at T=+4° C. is designated as ⁶⁴Zn—Z2 in charts andtables.

As a result of immunocytochemistry assay of cell antigens-cadherins andCD44 marker, significant changes in the studied proteins after theirexposure to the action of the components were detected. The effectsobserved in the course of the experiment differed depending on the dosesof the preparations, and ⁶⁴Zn—Z2 component showed increased activityafter its storage for 14 days at temperature+4° C.

The action of ⁶⁴Zn at a dose of 0.128 mcg/ml resulted in increasednumber of E-cadherin-positive cells (cells with E-cadherin expression)and a slight increase in N-cadherin positive cells. At a dose of 3.2mcg/ml, this material caused a decrease in the number ofE-cadherin-positive cells and a substantial increase inN-cadherin-positive cells and CD44 marker, as shown in FIGS. 45, 46, 47,48. This fact evidences blocking one of the pathogenic ways of the tumorgrowth—metastasis of tumor cells. An increase in the number ofE-cadherin-positive cells causes an increase in their adhesiveproperties and, as a consequence, reduction of their invasive andmigratory potential (which is characteristic for cells of the aggressivemesenchymal phenotype, with no or a small number of cells expressingE-cadherin).

Referring to the above, the use of the preparation at a dose of 3.2mcg/ml showed a decrease in the E-cadherin indicators and an increase inthe expression of CD44, a stem cell marker, which is indicative of“redistribution” of the adhesive properties from E-cadherin protein toCD44. Analysis of the slug transcription factor responsible for“aggressive” behavior of tumor cells also shows activity of tumor cellsand their characteristics in terms of epithelial-mesenchymal transition.This factor is a marker of mesenchymal (aggressive) cells. It isindicative of high migratory and invasive abilities of cells which andas a result of complication in the disease progression in cancerpatients.

The action of ⁶⁴Zn—Z2 component (⁶⁴Zn after 14 days storage at T=+4° C.)on the phenotype of PA tumor cells affects the number ofE-cadherin-positive cells for both doses of 3.2 mcg/ml and of 0.128mcg/ml. Their number grows, especially the number of cells and theexpression level that corresponds a dose of 0.128 mcg/ml as shown inFIGS. 45, 49. At the same time the N-cadherin level does not change. Thenumber of cells and their expression level are similar to those of thecontrol cells not exposed to the action of the active component, asshown in FIGS. 45, 60. Thus, a similar phenotypic changes, that weredetected under the action of ⁶⁴Zn, were also observed under the actionof ⁶⁴Zn—Z2.

The general criterion of similarity in the effects of sample materials⁶⁴Zn and ⁶⁴Zn—Z2 preparation is an increase in the number ofE-cadherin-positive cells at dose D1, and the maximum result—300 scoreson to the H-Score scale—was obtained with the use of ⁶⁴Zn—Z2 component.

The fact that cells are expressing CD 44 stem marker for the ⁶⁴Zn—Z2preparation at a dose of 0.128 mcg/ml is indicative of significantlyhigher adhesive properties of the cells as compared with a dose of 3.2mcg/ml. The values of CD44 marker positive cells at a dose of 3.2 mcg/mlfor ⁶⁴Zn and ⁶⁴Zn—Z2 are close with tabulated values of 126 and 118units on the H-Score scale.

The effect of ²⁴Mg in 2 doses was an increase in the number ofE-cadherin-positive cells and a slight increase in the number of cells,expressing N-cadherin. It indicates domination of epithelial (lessmalignant) characteristics in these cells after their treatment with²⁴Mg containing material. This results in inhibition of their migratoryand invasive properties confirmed by scratch assay and analysis oftranscription factors which are the key epithelial-mesenchymaltransition criteria. The latter are crucial in formation of anaggressive metastatic cell phenotype and plays a key role in themetastasis processes and in the course of a tumor process in general,since acquisition of metastatic and mesenchymal characteristics by cellsis indicative of the disease progression.

The number of CD44-positive cells after the action of ²⁴Mg at a dose of2 mg/ml did not differ from that of control cells. The dose of 3.5 mg/mlcauses their number to increase significantly. Besides, a clonaldifference in the expression of this protein, which is not only a stemcell marker but also a protein of adhesion and cytoskeleton, wasobserved in the cell population. Clones with the average number ofpositive cells and the average level of expression and the clones withmore higher characteristics were identified in FIGS. 45, 54.

The effect of ³⁹K on the PA is quite interesting. Dose dependence issignificant for this isotope. There is also a possible clonallyselective action since different phenotypic manifestations depend on thefield visualization were observed in cytospin preparations of cellsunder the effect of ³⁹K as shown in FIGS. 45, 56, 58. The ICC analysisshowed that the number of E-cadherin-positive cells significantlyincreased with the action of this preparation at a dose of 0.4 mg/ml andsignificantly reduced (up to almost complete inhibition) at a dose of 2mg/ml as shown in FIGS. 45, 56.

A similar tendency towards almost complete inhibition of anotheradhesion protein, N-cadherin, was observed under the action of ³⁹K atdose D2 as shown in FIGS. 45, 57. At the same time the number ofCD44-positive cells significantly increased with the action of materialsat both doses as shown in FIGS. 45, 58. The observed concentrationdifferences are decisive in clinics when choosing therapeutic dosages.Furthermore, they are potential modifiers of epithelial-mesenchymaltransition, as they produce a significant impact on the markers typicalfor the process which evidences the potential impact on metastaticproperties of tumor cells and, accordingly, on the process of metastasisin general. In addition, changes in the expression of the stem cellmarker were detected which is also an important element in thecharacterization of the inhibitory (slowing down the tumor developmentprocess) properties of the light isotopes containing materials.

Changes in the expression of markers of epithelial-mesenchymaltransition and stem cells are also associated with the sensitivity oftumor cells to therapies and various biologically active agents whichmakes tumor cells therapeutically more susceptible (open) to the actionof light isotope components or other drugs. This enables a comprehensiveapproach to the treatment of tumors: first component makes the cell moresusceptible to further exposure and the following component (lightisotope containing one or any other) acts in a much lower dose toachieve the ultimate goal.

A wide range of variations of the values of adhesion markers and stemcells marker characterizes the light isotopes containing materials asefficient modifiers of epithelial-mesenchymal transition with apossibility to control its direction. Increase in the expression levelof the adhesion molecules (E, N cadherins) after the action of lightisotopes containing materials on tumor cells of PA renal carcinomaevidences blockage for one of the main ways of neoplastic processdevelopment—metastasis.

The phenotypic characteristics of cells after exposure to lightisotope-containing materials are characterized by epithelial type, i.e.,low invasive and migratory potential, which reduces the severity ofcancer. ⁶⁴Zn—Z2 component after 14 days of storage showed the bestresult in terms of a number of cadherin-positive cells, assessed on theH-Score scale based on the ratio of the dose-result criteria (the numberof cadherin-positive cells). The dose of 0.128 mg/ml showed a score of300 points on the H-Score.

A comprehensive approach to anti-cancer and anti-tumor therapyconsisting of two or more steps can be used. At the first step, by using³⁹K (which provided the greatest increase in the number of cellspositive to cadherin and CD44-marker at dose D1-.4 mg/ml), we boost celladhesion to the maximum and facilitate enhancement of susceptibility ofcells to therapy, and at the second stage we act therapeutically on thepre-treated cells by using ⁶⁴Zn or ⁶⁴Zn—Z2 at dose D1-0.128 mcg/ml (asthis concentration is the most rational from the point of view of thedose-effect ratio).

The combined therapy is recommended in combination with surgicaltreatment: before and after surgical operation on the primary tumor(primary lump or focus) and will contribute to the prevention ofpossible metastases and occurrence of secondary foci.

Epithelial-mesenchymal transition and a transcription factor as criteriafor assessing phenotype in A-549, MCF-7 and COLO 205 human tumor cellsand normal rat cells (NRK) after their exposure to the action ofcomponents containing ³⁹K, ⁶⁴Zn and ²⁴Mg. Detection of the effect ofcumulation of antitumor properties of light isotopes containingmaterials on the A-549 cell line. The term “epithelial-mesenchymaltransition” (EMT) refers to the definition of the process ofreprogramming of epithelial cells which normally occurs during embryonicdevelopment or wound healing in the adult organism. Historically thedefinition of epithelial and mesenchymal cells was based on thedifference in their visual characteristics and morphology multicellularstructures they form. In particular, it was found that epithelial cellscreate strong intercellular contacts and form a dense monolayer, whichexcludes separation of individual cells. Mesenchymal cells, in contrast,do not form any ordered structures and do not create tight intercellularcontacts thereby reducing their adhesive properties and increasing theirability to migrate. The phenotype of cells can change under theinfluence of certain factors and cells can acquire the properties ofboth types.

Conversion of epithelial cells (less malignant) into mesenchymal (withgreater malignancy) is accompanied by changes in their morphology,adhesion and migration ability. There are a lot of markers, thatcharacterize these types of cells at the molecular level. Standardchanges during EMT include increased expression of N-cadherin andvimentin, nuclear localization of β-catenin and increased expression ofsuch transcription factors as Slug, Twist and E47, which inhibit theexpression of E-cadherin, which in its turn, causes an increasedcapacity of cells for migration and invasion as well and associated withtheir resistance to apoptosis.

Thus, in result of EMT in the tumor progression, benign tumor cellsacquire capacity for invasion and migration, which in its turn, leads todissemination of tumors and development of the malignant process. Inaddition, tumor cells with a predominance of mesenchymal characteristicsare less sensitive to anticancer drugs and therefore have a resistantphenotype.

In recent years, we find more and more data about the interrelation of“stem tumor cells” and EMT, in particular. It is reported that thesecells appear partly due to EMT. There are data that mesenchymal cellswith metastatic phenotype are characterized by the properties of stemcells, namely CD44+CD24− (cells of human breast cancer), and stem cellsisolated from normal or tumor breast tissues express some standard EMTmarkers. That is why a comprehensive study of EMT protein markers,transcription factor and stem cell antigens in tumor cells of adifferent histogenesis after their exposure to the action ofpreparations under investigation has become the subject matter of thispaper.

Cells of human non-small cell lung cancer (line A-549), human breastcancer (MCF-7 line), human colon adenocarcinoma (COLO 205 line) andnormal rat kidney cells (NRK line) (derived from the Bank of Cell Linesfrom human and animal tissues, R. E. Kavetsky Institute of ExperimentalPathology, Oncology and Radiobiology, National Academy of Sciences ofUkraine) were cultured in complete culture medium RPMI 1640 (PAA)/DMEM(PAA), supplemented with 10% newborn/fetal calf serum (depending on thecell type) (PAA) and incubated in 5% CO₂ humidified atmosphere at 37° C.The culture medium was replaced in 2 to 3 days.

When making the immunocytochemistry (ICC) assay, cells on microscopeglasses (cytospin preparations) were fixed in the solution(methanol+acetone 1:1) for 2 hours at t 20° C. and incubated with 1%solution of bovine serum albumin (BSA) for 20 minutes. Then themonoclonal antibodies-CD44 (Diagnostic BioSystems), E-cadherin(ThermoScientific), N-cadherin (NeoMarker, BioLegend), SLUG(GeneTex)—were applied for the time period specified by the manufacturer(30 to 60 minutes), after which the Poly Vue imaging system conjugatedwith peroxidase was used and the enzyme activity was detected usingdiaminobenzidine (ThermoScientific) as a substrate. After theimmunocytochemical reaction the preparations were washed with water andcounterstained with hematoxylin-eosin (for 15 to 30 seconds).

The results were analyzed by counting cells with expression (browncolored cells) using a light microscope, and were assessed using theclassical H-Score method: S=1×A+2×B+3×C, where S is the H-Score indexgiving a range of 0 (protein is not detected) to 300 (high-levelexpression in 100% of the cells); A is the percentage of weakly stainingcells, B is the percentage of moderately staining cells and C is thepercentage of strongly staining cells.

Statistical processing of the results was performed using a mathematicalbiomedical program STATISTISA 6.0. Calculation and comparison of thesignificance of differences of mean values were performed using theStudent's t-test. In our study into antigens associated withepithelial-mesenchymal transition and stem characteristics of tumorcells, we examined cadherins-adhesion and cytoskeleton proteins, CD44and Slug transcription factor after their exposure to the action of⁶⁴Zn, ²⁴Mg and ³⁹K containing materials. We studied three types ofcells. Cells of human breast cancer-MCF-7 line characterized byepithelial (less malignant) phenotype (i.e., epithelial markers dominatein cell antigens of this line), cells of human non-small cell lungcancer-A 549 line characterized by a mixed phenotype (with markers ofboth mesenchymal and epithelial cells) and cells of human colonadenocarcinoma-COLO 205 line in which mesenchymal (more malignant)markers dominate.

It has been found that the light isotopes produced the greatest effecton the adhesion protein—E-cadherin—which is a characteristic marker ofepithelial cells and its presence is indicative of a less aggressivecellular behavior. The action of ³⁹K component resulted in inhibition ofa number of cells with the given antigen in the A-549 line, whereas itsignificantly increased the number of E-cadherin-positive cells in themesenchymal COLO 205 line which evidences its potential selective effecton cells with a more aggressive mesenchymal phenotype, as well asinhibition of the latter. At the same time, this component acted onanother cytoskeletal protein, N-Cadherin, which is a marker ofmesenchymal cells, as well as on stem cell marker CD44. The action of⁶⁴Zn component was characterized by the following results: the number ofE-cadherin-positive cells significantly increased in the line with amore aggressive mesenchymal phenotype. Cells with less malignantpotential (MCF-7 and A-549) showed smaller values of expression whichalso characterizes ⁶⁴Zn as a component with a pronounced selectiveaction directed at cells with the mesenchymal (malignant) phenotype.These facts give evidence of strong effects of ⁶⁴Zn on adhesion andcytoskeletal proteins that affect their “malignancy” characteristics aswell.

⁶⁴Zn component virtually had no effect on the expression of CD44 stemcell marker, although interesting was the fact of its intracellularredistribution. This antigen was expressed very specifically and indifferent ways after the action of ⁶⁴Zn, ²⁴Mg and ³⁹K components. Minorquantitative changes in the components led to redistribution of itsexpression from the nucleus to the peripheral areas. ²⁴Mg component alsoquite effectively influenced the cells with a more aggressivemesenchymal phenotype (COLO 205) by increasing the number ofE-cadherin-positive cells which characterizes its role as an inhibitorof the mesenchymal phenotype.

The data obtained in the study of antigens of immortalized(conditionally normal) cells of a rat (NRK) show that the phenotype ofnormal cells has not changed after their exposure to the action of ⁶⁴Znand ⁶⁴Zn—Z2 components (which were the most effective in tests both forcancer and for these cells). i.e., these components did not cause anychanges in normal cells NRK which is a positive characteristic of thecomponents and indicates that normal cells NRK retain their phenotypicstability. The fact of elocalization (redistribution) of antigens ofN-cadherin and CD44 has been observed, which is a very interesting factand requires further more detailed studies.

In the study of a slug transcription factor, which according to theliterary data is a marker of mesenchymal (malignant) phenotype of cellsand shows an aggressive metastatic phenotype of tumor cells, interestingfacts have been found. Thus, ³⁹K, ⁶⁴Zn and ²⁴Mg components virtually didnot influence manifestation of this factor in cells with epithelial(less aggressive) phenotype.

The preparations had multidirectional effects on cells with a mixedphenotype (A-549) with an observed relocalization of the factor from thenucleus to the cytoplasm and vice versa. The number of slug-positivecells with nuclear expression significantly decreased after the actionof ³⁹K component, while after the action of ⁶⁴Zn component the decreasein cells with cytoplasmic expression was observed.

In the study of slug transcription factor in COLO 205 cells with themesenchymal phenotype, ³⁹K component increased its expression in thecytoplasm, and ⁶⁴Zn and ²⁴Mg components—in the cytoplasm and in thenucleus. Thus it is known from the literature that Slug is a suppressorof E-cadherin, i.e. when it is activated, the number ofE-cadherin-positive cells should fall, but we observed a differentpicture—their number increased, which is an evidence of activation ofthe epithelial (less malignant) cell phenotype.

Alluding to the above, the effect of accumulation of antitumorproperties of light isotopes containing materials on A-549 tumor cellline will now be discussed. When working with the preparations understudy, it was noted that the functional activity of certain doses of thecomponents increased over time after preparation of the solution and itssubsequent storage at t+4° C. This phenomenon was detected on the A-549cell line (human lung cancer). Activity of ⁶⁴Zn relative to its abilityto transform cells from primary tumor cells into cells of type A(similar to stem cells) and of type B (no tumor) differed over time.FIGS. 11 and 12 show the results of quantitative analysis of cells (%)versus control (no preparations) after the action of substances at adose of 16 mcg/ml dissolved at different times (FIG. 11): 60 days beforethe experiment, 28 days before the experiment and immediately before theexperiment. The preparation was stored at the temperature of +40° C.

Changes in the functional activity of ³⁹K sulfate form at a dose of 2mg/ml are shown in FIG. 91. After 20 days of storage of the preparationat T+4° C. the above mentioned dose showed an increase in the componentfunctional activity expressed in a larger number of detected cells oftype A. Multiple doses that differed 5 times upward and downward (10mg/ml and 0.4 mg/ml) did not confirm such effect. ³⁹K sulfate form at adose of 10 mg/ml showed no presence of cells of type A on the first andon the 20th day of the experiment, and the dose of 0.4 mg/ml showedidentical (in terms of quantity of A cells) results.

Cytogenetic characteristics of A-549, MCF-7 and COLO-205 cell linesafter their exposure to the action of materials containing ³⁹K, ⁶⁴Zn and²⁴Mg will now be discussed. Advanced Micronucleus Test was used toassess the cytogenetic characteristics of cells after their exposure tothe action of the test group of components. The effects of componentscontaining ³⁹K, ⁶⁴Zn and ²⁴Mg on A-549, COLO-205 and MCF-7 tumor celllines were assessed both by the number of micronuclei (by fragments ofchromosomes or remained chromosomes) and by morphologicalcharacteristics, such as internuclear bridges (dicentric chromosomes),nuclear protrusions (gene amplification) (Fenech, 2002) and apoptoticcells.

The effects of materials containing ³⁹K, ⁶⁴Zn and ²⁴Mg were assessed atthe molecular, cellular and chromosomal level depending on the dosage ofthese components. Cytogenetic analysis was carried out after 48 hours ofexposure of the cells to the action of the components. For cytogeneticanalysis, the cells were incubated in hypotonic solution KCl (0.54%)(Reahim, Ukraine) for 40 minutes at +37° C. Then they were fixed with amixture of methyl alcohol (Reahim, Ukraine) and acetic acid(Himlaborreaktiv, Ukraine) (3:1) with replacing the fixative threetimes. All fixed cell suspensions were applied on cold wet glasses,dried and stained with Giemsa stain (Sigma, Germany). The cytogeneticpreparations were analyzed using a binocular microscope Sari Zeiss,AxioStarPlus (Germany) with magnification of x 1000. The followingquantitative characteristics and stages of cell division were analyzedon the preparations: mitoses, cells with premature chromosomecondensation (PCC), cells with micronuclei (CMN), apoptotic cells andcells with nuclear protrusions (protrusions) which were calculated for1000 cells and expressed in per mille (%).

Doses of the components used and cytogenetic characteristics were ³⁹K ata dose of 2 mg/ml, ⁶⁴Zn at a dose of 20 mcg/ml and ²⁴Mg at a dose of 4mg/ml cause inhibition of tumor proliferation (cell division) in thecells of COLO 205 line at the stage of G2/M and significantly increasethe number of nuclear shape anomalies. The detected increase in thenumber of apoptotic cells and protrusions while maintaining the numberof micronuclei on the COLO 205 cell line is shown in as shown in FIG.99.

Formation of the largest number of nuclear protrusions was recorded forA-549 cell line.

The maximal effect was observed after the use of the componentcontaining ³⁹K at a dose of 2 mg/ml. The effect produced by this doseconsisted in slowing down proliferation of the tumor cells, formation ofmicronuclei and various anomalies of the nuclear shape, including shapesof segmented cell destruction. The action of ⁶⁴Zn component also led toan increase in micronuclei and nuclear protrusions in comparison withthe control cells. The action of ⁶⁴Zn at a dose of 20 mcg/ml caused atriple increase in the level of nuclear abnormalities as compared to thedose of 10 mcg/ml and fifty times as high as the baseline (control). Alarge number of damaged cells of non-apoptotic type, shown in FIG. 100,was observed. The action of ²⁴Mg was also characterized by accumulationof nuclear anomalies and weakening of the proliferative ability of tumorcells. An increase in the number of apoptotic cells was observed afterthe action of all three isotopes.

Formation of nuclear protrusions was observed in the cells of MCF-7 linebut in an amount smaller than in the previous experiments. The action of⁶⁴Zn in this cell line causes more than a two-fold increase in the levelof cells with micronuclei as shown in FIG. 101. An increase in thenumber of apoptotic cells compared to the control ones was detected.

Dose-dependent effects of the isotopes on various cell lines wererepresented as quantitative characteristics of the degree of abnormalityof nuclei. The graphs in FIG. 102 show the quantitative characteristicsof nuclear structure abnormalities, such as apoptosis, protrusion andcells with micronuclei. The results are compared with the control groupthat was not exposed to the isotopes containing materials. The maximumvalue of the main cell anomalies such as protrusions and apoptosis wasrecorded on the A-549 cell line for ³⁹K at a dose of 2 mg/ml and for⁶⁴Zn at 2 doses, while the maximum number of protrusions and cells withmicronuclei was observed after the action of ³⁹K at a dose of 2 mg/mlwhich is also characterized by reduction of tumor cell proliferation.

Given the similarity of the data about nuclear anomalies (which areexpressed in a similar character of cellular abnormalities in all celllines), one can conclude that a significant number of micronuclei formedin result of nuclear protrusions. Disruptions of the integrity of thenucleus such that the cell loses its ability to further functioning isassociated with the beginning of nucleus destruction as shown in FIG.98.

Major differences in sensitivity of the cell lines were as follows. TheCOLO 205 cell line showed sensitivity to all the preparations by anincrease in the number of cells with abnormal shape of the nucleus,while sensitivity of the MCF-7 line was expressed in a smaller extentand was characterized mainly by the presence of chromosomal aberrations.These disorders are characterized by fragmentation of nuclei in theeukaryotic cell that does not contain the full genome needed for itssurvival. The component with ²⁴Mg on the MCF-7 cell line leads toaneugenic disorders (not associated with the structural damage ofchromosomes).

It has been established that all the light isotopes containing materialswith ⁶⁴Zn, ²⁴Mg or ³⁹K—had the maximal effect on E-cadherin, the markerof epithelial cells, in COLO 205 cells with domination of mesenchymalcharacteristics. A selective nature of the action of all light isotopeshas been shown. An increase in the expression of E-cadherin to themaximum extent occurred on the COLO 205 cell line. The phenotype ofthese cells was the most malignant of the 3 human cell lines involved inthe experiment, namely A-549—non-small cell lung cancer, MCF-7—breastcancer and COLO 205—colon adenocarcinoma. Thus, the effect of theinvestigated materials was directed to the most malignant cells, and theexpression of epithelial E-cadherin indicates inhibition of themalignant properties of cells after their exposure to the action of saidmaterials.

Alluding to the above, ³⁹K containing material has shown the bestability to inhibit the mesenchymal (cancerous) phenotype (score 184 onthe H-Score scale compared to 87 in the control). This isotopeinfluenced the COLO 205 cells with most aggressive mesenchymal phenotypeby significantly increasing the number of E-cadherin-positive cellsconfirming its role as an inhibitor of the mesenchymal phenotype oftumor cells.

The effects of the materials on CD44 stem cell marker were expressedmainly by its intracellular redistribution. This antigen has not shownany regularity relative to the dose used or cell type. No significantchanges in the expression of this marker has been found in all the testtumor cell lines. It has been shown that ⁶⁴Zn, ²⁴Mg and ³⁹K isotopesinsignificantly influenced the number of Slug-positive cells (with thetranscription factor—a marker of mesenchymal phenotype) in the MCF-7line with epithelial (less aggressive) phenotype. Their effects on thecells with the lowest malignant potential (of 3 tumor cell linesinvolved in the experiment) were minimal. The effects of the isotopes onthe cells with a mixed phenotype (A-549) were manifested inrelocalization of the Slug factor from the nucleus to the cytoplasm andvice versa.

The number of Slug-positive cells with nuclear expression significantlydecreased after their exposure to ³⁹K component, while the action of⁶⁴Zn resulted in decrease in the number of cells with cytoplasmicexpression. The highest expression of Slug transcriptional marker hasbeen observed in the most malignant cell line′COLO 205, which indicatesthat ⁶⁴Zn, ²⁴Mg and ³⁹K isotopes activate an active phase of theepithelial-mesenchymal transition program.

The selective nature of increasing expression of E-cadherin has beenfound being highest on the COLO 205 cell line and is characterized by anincreased adhesion to the matrix. It indicates that the cellularphenotype is directed towards lower malignancy and that the action ofthe isotopes causes inhibition of malignant properties of cells.

It has been established that ³⁹K component increases the expression ofSlug transcription factor in the cytoplasm and ⁶⁴Zn and ²⁴Mgcomponents-both in the nucleus and in the cytoplasm in COLO 205 cellswith mesenchymal phenotype, which in addition to modification of theirphenotype towards lower malignancy also indicates a change in theirsensitivity to the antitumor agents.

According to the new data in the literature, the transcription factorunder study indicates the activation of mesenchymal phenotype. With itsstable over-expression an increase in the sensitivity of tumor cells toantitumor agents which are targeted at microtubes of the cellularcytoskeleton and tubulin were observed. This allows to perform givesanti-tumor therapy in two stages. First stage would consist in theaction of one of the components on the cell line to increase itssensitivity to anticancer agents. The second stage would consist in theanticancer therapy with reduced dosage due to the high sensitivity ofthe cells to the action of the administered drugs.

All three isotopes have strong selective effect on the most malignant(mesenchymal) cells. Slug, a marker of malignancy, and cadherins,markers of adhesion, have shown the highest expression in themesenchymal cell line-COLO 205. The action of all the tested materialson normal rat cells (NRK) did not resulted in any changes in theirphenotype. It indicates that normal cells retain their phenotypicstability under the effect of light isotopes.

A cumulative antitumor effect of the ⁶⁴Zn and ³⁹K sulfate form has beenshown on the example of A-549 cell line. The action of ⁶⁴Zn caused anincrease in the number of transformed cells from the primary tumor tocells of type A with the storage of the ⁶⁴Zn sulfate form solution for60 days. After this period, the number of A cells after the use of ⁶⁴Znincreased up to about 80% compared with 20% recorded at the initialstage of the experiment. A similar result was also found for the ³⁹Ksulphate form at a dose of 2 mg/ml on the A-549 cell line. The number ofcells of type A (after 20 days of storage of the solution) was 2-foldhigher than on the first day of control. It should be noted that theobserved cumulative property of ³⁹K is dose-dependent at it did notmanifest itself at 5-fold doses (upward or downward). This property wasnot manifested at 10 mg/ml and 0.4 mg/ml. 100% transformation of theinitial tumor cells to the cells of type A was shown at a dose of 0.4mg/ml (in both cases), and the dose of 10 mg/ml was not effective and notransformational effect was found.

The action of materials containing isotopes ³⁹K, ⁶⁴Zn and ²⁴Mg on humantumor cells leads to dose-dependent effects which consist in theformation of a large variety of abnormalities of the nucleus, such asprotrusions, apoptotic corpuscles and micronuclei. For all the isotopesunder study a massive accumulation of cells with protrusions wasrecorded. Their 15-fold increase under the action of ³⁹K component andalmost 10-fold increase under the action of ⁶⁴Zn component were observedin the A-549 cell line. A positive correlation between the formation ofmicronuclei, nuclear protrusions and nucleoplasmic bridges associatedwith the instability of the genome and gene amplification by initiationof breaking-fusion-bridge cycles was observed under deficiency of folicacid as well as under ionizing radiation used in the radiotherapytreatment of cancer patients.

Processing of cells of the COLO-205 and A-549 cell lines with ³⁹K, ⁶⁴Znand ²⁴Mg isotopes resulted in accumulation of cells with prematurechromosome condensation. An increase in their number relative to cellsin mitosis is indicative of the cell cycle arrest at the G2/M phase.

Prolonged mitotic arrest usually leads to DNA damage and induction ofp53 and then to apoptosis. A positive correlation between G2/M-arrestand induction of apoptosis was detected in human ovarian cancer cellsafter their exposure to radiation. Thus, the arrest of the cell cycle inthe G2/M phase is an important stage on the way from the genotoxiceffects, causing DNA damage, to apoptosis.

The action of ³⁹K isotopes causes maximal (in comparison with othercomponents) inhibition of cell division at the G2/M stage of the cellcycle. It is known, that the action of some anticancer drugs, such aspaclitaxel, is also associated with the phase of premature chromosomecondensation. In the case of paclitaxel, its action directed to theinduction of apoptosis and the expression of ligands on the cellsurface, which in their turn attract K cells for the removal of damagedcells. The effects of ³⁹K, ⁶⁴Zn and ²⁴Mg isotopes on all cell lines werecharacterized by an increase in concentration of cells with micronucleiof clastogenic and aneugenic origin.

The A-549 cell line (human lung cancer) has turned to be the mostsensitive to all the components under study. Assessment of the effectsof ³⁹K, ⁶⁴Zn and ²⁴Mg on human tumor cells was obtained after 48 hoursof interaction of the components with cells. During this time no morethan two full cycles of cell division passed. Therefore, the results ofthe effects of the light isotopes (quantitative characteristics ofnuclear abnormalities and proliferative capacity of cells) must beconsidered in relation to this circumstance. The effects of the isotopeson tumor cells depend on the stage of the cell cycle, and they manifestitself differently at various stages of the cycle. By increasing thetime of interaction of ³⁹K, ⁶⁴Zn and ²⁴Mg with the cell population, weincrease the number of contact points in the component cell system (interms of the full cell cycle) and the number of full cell cycles duringwhich the interaction of the components with cells occurs.

The presence of apoptotic cells (found in a smaller number as comparedwith protrusions) is associated with a full cycle of cell divisionduring which the isotopes exerted their action during the whole phase ofthe cell cycle. The presence of protrusions evidences the action of theisotopes, which started at a stage other than the initial stage of celldivision. Since the number of cells in the population being at a stagedifferent from the initial stage of division was much larger than thecells at the initial stage, then, accordingly, the number of cells withprotrusions was much larger than the cells with apoptosis.

The presence of a sufficiently large number of anomalies in thestructure of the cell nucleus which were expressed in the form ofprotrusions, as well as their character, confirms the presence ofclassic chromosomal abnormalities (damage of the DNA structure) which inmost cases results in the loss of the capacity of cells for replication(doubling) the DNA helix. A similar pattern (in terms of loss of thecapacity of cells for further division and multiplication) is observedin the case of programmed cell death-apoptosis. Thus we can concludethat the action of ³⁹K, ⁶⁴Zn and ²⁴Mg isotopes on human tumor cellsanalyzed within 1-2 cycles of cell division causes the launch of themechanism of cell death similar to apoptosis (by the end result—the lossof capacity for further division).

The cell capacity for apoptosis is its natural function and the lack ofit is one of the basic features of tumor cells. The results ofcytogenetic analysis of tumor cells after their exposure to the isotopesof ³⁹K, ⁶⁴Zn and ²⁴Mg shows the restoration of a limited number ofcycles of cell division which is typical for normal cells.

Comparative characteristics of the action of Doxorubicin and the actionof components containing ³⁹K, ⁶⁴Zn and ²⁴Mg on normal rat kidney cells(NRK) and comparative assessment of the effects of ³⁹K, ⁶⁴Zn and ²⁴Mg vsDoxorubicin on normal rat kidney cells (NRK cell line) will now bediscussed. After the data about the effects of ⁶⁴Zn, ²⁴Mg and ³⁹Kcomponents on tumor cells of MCF-7 (human breast cancer), A-549 (humanlung cancer) and COLO 205 (human colon adenocarcinoma) cell lines havebeen obtained, an experiment was conducted to determine the effects of⁶⁴Zn and ³⁹K components on normal/immortalized rat kidney cells NRK. Animmortalized cell line is a stable, capable of unlimited proliferationcell line that consists of cells with a limited lifetime in culture. Theaction of freshly prepared solutions containing ⁶⁴Zn and ³⁹K isotopeswas tested on NRK cells in comparison with doxorubicin as shown in FIG.103.

0.9% NaCl solution with 5% glucose was used as the solvent for ⁶⁴Zn andK. The obtained data and images show that the effects of ⁶⁴Zn and ³⁹Kisotopes were associated with the transition of initial NRK cells intocells similar to type A, and the action of anticancer drug Doxorubicin(in concentrations ranging from 400 to 0.01 mcg/ml) shows the number oflive and dead cells.

Effect of chemotherapeutic drug Doxorubicin at doses ranging from 400 to16 mcg/ml during 48 hours on the normal NRK cells resulted in the lossof their mobility, ability to proliferate and complete death. ⁶⁴Zn and²⁴Mg sulfates at the same concentrations as used for doxorubicine didnot affect functional properties of RNK cells. Doxorubicin's ability toinhibit the natural biological properties of normal cells until theirdeath considered as side effect. It significantly reduces its use andconcentration range of administration.

In the concentration range from 3 to 0.01 mcg/ml Doxorubicin produces aside effect causing death of normal NRK cells. Reduction in theconcentration of doxorubicin increased the number of viable cells. Theeffects of the sulfate form of ⁶⁴Zn and ²⁴Mg isotopes on the A-549 tumorcell line show signs of similarity of the A-549 cells treated with ⁶⁴Znand ³⁹K with stem cells of a mouse.

Assessment of migration characteristics of A-549, FC, NRK, HaCaT, A-431and MM-4 cell lines after their exposure to the materials containing³⁹K, ⁶⁴Zn and ²⁴Mg. Analysis of the combined effects of isotopes of ³⁹K,⁶⁴Zn and ²⁴Mg and Doxorubicin will now be discussed.

Understanding the biological mechanisms of tumor cell migration andsearch for potential “anti-migration” drugs is an important issue ofmodem oncology since the cell migration activity is one of the keycharacteristics of malignancy and an important stage of their metastaticpotential.

Today there is no more doubt in the importance of the role ofepithelial-mesenchymal transition (EMT) as the main regulator of themetastatic cascade [Raghu Kalluri, Robert A. Weinberg. The basics ofepithelial-mesenchymal transition. J. Clin. Invest 2009; 119: 1420-1428,Samy Lamouille, Jian Xu, Rik Derynck. Molecular mechanisms ofepithelial-mesenchymal transition. Nature Reviews Molecular Cell Biology2014; 15: 178-196.]. In modern oncology it is common practice todistinguish steps of the metastatic cascade such as loss of celladhesion, abnormal motility, invasion, intravasation, survival in thecirculation, extravasation, metastatic colonization, and treatment ofclinically diagnosed metastasis.

Heterotypic interactions of epithelial and mesenchymal cells arenecessary for normal morphogenesis at all stages of embryonicdevelopment. During carcinogenesis such interactions provide increasedmalignant phenotype of cancer cells. The cardinal feature of anepithelial cancer cell is its abnormal mobility and ability to separateand penetrate into the surrounding tissues, i.e., to invasion. Comparedwith normal epithelium carcinoma, cells are characterized by aprogressive decrease in cell adhesion and increase in their migrationabilities. In the clinical oncology such notions as “the tumor surface”and “invasive front” of the tumor acquire principal importance asmalignant cells leave the primary tumor and enter the blood flow justfrom the surface of the tumor, from its leading edge. It is at theleading edge where the tumor cells are transformed from epithelial tomesenchymal ones [Kovalev A. A. Metastatic cascade as a therapeutictarget. Health of Ukraine.—2011: p. 26-28].

According to the latest data of R. Weinberg (2010), cells in theepithelial-mesenchymal transition state acquire properties of cancerstem cells with the implementation of their main functions-metastasis,colonization and the ability of division in distant organs as well asthe possibility of colonization of the primary tumor and stimulation ofits growth (self-seeding hypothesis). Many biological processes of tumorgrowth and metastasis as well as cases of drug resistance and tumorrecurrence after treatment are associated with the phenomenon ofepithelial-mesenchymal transformation.

Modem approaches in fundamental and clinical studies of the recent yearsshow a clear shift of the scientific interests towards the study of themetastatic properties of cancer cells.

According to the generalized statistical data, only 0.1% of circulatingtumor cells are able to form metastatic lesions in distant organsshowing their invasive properties, and because in most cases in clinicalpractice a patient's death occurs in connection with an increase in thenumber of tumor cells, it is the invasive-metastatic cascade thatdetermines the progression of cancer and eventually causes the death ofa cancer patient.

Thus the tumor as a heterogeneous biological system that determines thefate of a patient is characterized by a combination of 2 majorproperties: invasion ability or invasion inability of 0.1% of the tumorcells that left the primary lesion. A vast majority of the remainingcells that left the tumor and died in the systemic circulation throughthe mechanism of apoptosis or returned to the primary tumor are not akey factor that causes the loss by any organ of its functions or thedeath of the body.

The rapid and uncontrolled growth of tumor cells with an unlimitednumber of divisions demonstrates the ability of tumor cells to overcomeanatomical and functional barriers formed over millions of years of thebiological evolution. According to Charles Darwin, the founder of theTheory of Evolution, it is not the strongest species that survive butthose that can better adapt to changing environmental conditions.Understanding the role of each of the participants in this process interms of genome constancy and chemical bonds that define theimplementation of the principle of interaction of circulating tumorcells with the cells of the vascular system (endothelial cells,dendritic cells, macrophages) will make it possible to create“anti-metastatic” drugs of new generation with a possibility toselectively regulate adhesion of cancerous cells in the bloodstreamwithout violating the integrity of physiological hemostasis, i.e.,enhance the interaction of tumor cells with the main lesion and reducethe possibility of invasion by circulating cells.

With the progression of a tumor disease doctors are often faced with thetumor resistance to anti-cancer therapeutic agents wherein the cellswith low sensitivity are characterized by to most aggressive phenotype.According to the researchers' data there are facts that prove selectionof the most aggressive cells with dominating mesenchymal characteristicsand, accordingly, increase in their capacity for migration and invasion,which contributes to their dissemination and formation of new metastaticlesions after the selective effects of antitumor drugs.

Therefore, the aim was to study the characteristics of migration oftumor cells under the effect of the well-known anti-cancer drugs and thetest substances which characterizes them as modifiers of cellularbehavior and potential regulators of cell migration which is animportant feature of the metastatic potential of malignant cells. Wealso paid attention to the effect of the test substances on normal cellsfor the purpose of their comparative potentially damaging, or vice versarestoring (regenerating), means in therapy.

The most illustrative and effective method for analyzing the migrationrate of tumor cells in vitro is the scratch assay which has severaladvantages. First of all, the migration of cells in vitro most fullyreflects the behavior of cells under in vivo conditions. Furthermore,this method can be used to study the mechanisms of regulation of cellmigration during the interaction of cells between themselves and withthe intercellular matrix [Liang Ch, Park, A Y, Guan, J L In vitroscratch assay: a convenient and inexpensive method for analysis of cellmigration in vitro. Nat Protoc 2007; 2: 329-33.].

Alluding to the above, materials, methods, and cell culture will now bediscussed. The cells of A-549, A-431, NRK, HaCaT, MM-4, normalfibroblasts (derived from the Bank of Cell Lines from human and animaltissues, R. E. Kavetsky Institute of Experimental Pathology, Oncologyand Radiobiology, National Academy of Sciences of Ukraine) were culturedin complete culture medium RPMI 1640/DMEM (PAA, Austria) (depending onthe cell type) supplemented with 10% fetal calf serum (PAA, Austria) andincubated in 5% CO₂ humidified atmosphere at 37° C. The culture mediumwas replaced in 2 to 3 days and the cells were passaged in 4 to 5 days.

Analysis of the influence of the components under study on the cellmigration rate was performed using the scratch assay method. Cells(5×104/well) were seeded into the wells of a 12-well plate in 2 repeatsin RPMI-1640/DMEM culture medium supplemented with 10% PBS, 40 mcg/ml ofgentamicin and incubated in 5% CO₂ humidified atmosphere at 37° C. for24 hours.

Then the components under study in different doses (from IC25 to IC50previously defined as a result of tests on verification of theantiproliferative effects of the components in vitro) and anti-cancerdrug Doxorubicin were placed in the respective wells. The plate wasincubated in a CO₂ incubator for additional 48 hours. After the cellsformed a dense monolayer it was damaged (by making a “scratch”) and thegrowth medium was replaced to remove cell debris and smooth the edges ofthe “scratch.” The rate of cell migration was analyzed using an invertedmicroscope and by photographing cells in the area of injury. Themigration activity of cells was fixed at several time points (dependingon the cell line analyzed). The microphotomicrographs were analyzed andthe cell incubation time needed to restore the monolayer was determinedfor all experimental groups.

At the first stage of the experiment an assessment of the effects of³⁹K, ⁶⁴Zn and ²⁴Mg isotopes on the nature of migration characteristicsof tumor cells of the human non-small cell lung cancer of A-549 cellline, immortalized rat kidney cells of NRK cell line and normal ratfibroblasts of RF cell line was carried out. This comparison was madenot only for the actual analysis of the potential anti-migration or, onthe contrary, restoration effects of the substances under study on cellsin vitro but also for the effects of these substances on normal andtransformed cells (immortalized or malignant).

A comparative analysis of the photos shown in as shown in FIGS. 113,114, 115, and 116 characterizes the migration activity of tumor cells ofthe human non-small cell lung cancer of A-549 cell line in the in vitroexperiment using the scratch assay and makes it possible to assess theeffect of several doses of ³⁹K, ⁶⁴Zn and ²⁴Mg isotopes on the process ofmigration of tumor cells. The following results were obtained after 72hours of observations of the migration activity of the cells.

All the light isotopes under study, ³⁹K, ⁶⁴Zn and ²⁴Mg, have shown theability to slow down the migration ability of tumor cells of the humannon-small cell lung cancer of A-549 cell line. The effects of thecomponents under study were assessed by comparing the time ofrestoration of the monolayer on the control group of cells, on the groupof cells exposed to the action of ³⁹K, ⁶⁴Zn and ²⁴Mg components and onthe cells exposed to the action of anti-cancer drug Doxorubicin.

A comparative assessment of the effects of antitumor Doxorubicin ascompared to the control group of cells showed that the use of the latterleads to a reduction in the time period of closing up the control gapthereby accelerating the process of migration of tumor cells.

The effects of different doses of ³⁹K, ⁶⁴Zn and ²⁴Mg components had thefollowing features. ⁶⁴Zn preparation at doses of 20 and 30 mcg/ml asshown in FIGS. 114, 115, 116E, F and ³⁹K preparation at a dose of 2mg/ml as shown in FIGS. 114, 115, 116C, D showed the best degree ofsuppression of the cell migration ability in the experiment with the A549 tumor cell line. Cells processed with these components in thespecified dosages, after 72 hours of their exposure to the action of thepreparations under study, showed incomplete (as compared to the control)restoration of the cell monolayer while the control cell monolayer wascompletely restored already after 24 hours following its damage.

For ⁶⁴Zn and ²⁴Mg components a direct dose-dependent concentrationeffect was observed and for light isotope ³⁹K at the doses of 2 mg/mland 3 mg/ml—a counter effect. ³⁹K component at the dose of 2 mg/mlshowed better ability to suppress the tumor cell migration activity ascompared to the dose of 3 mg/ml. The effects of both doses are shown inFIGS. 114, 115, 116C, D at various time intervals of the effects of thepreparations.

Anti-cancer drug Doxorubicin showed full restoration of the cellmonolayer after 24 hours following the beginning of its action, and atthe initial stage of observations (3 hours after the start of theexperiment as shown in FIG. 113-B its activity was higher as comparedwith the control group of cells. This characteristic suggests aconclusion about strengthening of the metastatic potential of tumorcells under the effect of even small doses of Doxorubicin.

According to the collective assessment of the observations made within72 hours, ³⁹K at a dose of 2 mg/ml and ⁶⁴Zn at the doses of 20 and 30mcg/ml showed the best results in terms of slowing the migrationactivity of tumor cells of human non-small cell lung cancer of A-549cell line.

Analysis of the data for the migration activity of A-549 cells showedthat ⁶⁴Zn preparation at a dose of 20 mg/ml as shown in FIGS. 118, 119Dand at a dose of 10 mcg/ml as shown in FIGS. 118, 119E suppresses cellmigration of the A 549 line. Cells processed with ⁶⁴Zn preparation at adose of 20 mcg/ml restored the cell monolayer in 72 hours after itsdamage and those treated with a smaller dose of the same preparation (10mcg/ml—in approximately 60 hours, whereas the cell monolayer in thecontrol group was completely restored 24 hours after damage.

Treatment of cells with the reference drug Doxorubicin at a dose of 0.2mcg/ml as shown in FIG. 118-B leads to an increase in the migration rateof the cells: restoration of the cell monolayer is observed earlier than24 hours following its damage. When culturing A-549 cells in thepresence of a combination of ⁶⁴Zn (10 mcg/ml) and Doxorubicin (0.02mcg/ml), as well as in the groups where the cells were treated only with⁶⁴Zn at a dose of 10 mcg/ml or only with Doxorubicin at a dose 0.02mcg/ml, suppression of the migration activity of tumor cells wasobserved, i.e., the cell monolayer damages were restored in 60 hoursfollowing the start of the experiment as shown in FIGS. 118, 119F.

Treatment of A-549 cells with a combination of ⁶⁴Zn at a dose of 20mcg/ml and Doxorubicin at a dose of 0.02 mcg/ml as well as the treatmentof these cells with ⁶⁴Zn alone at a dose of 20 mcg/ml causes asignificant suppression of the cell migration activity. In this case thecells restore the damaged monolayer in 72 hours after the damage tookplace as shown in FIG. 118, 119E.

Assessment of the effect of ³⁹K, ⁶⁴Zn and ²⁴Mg on migrationcharacteristics of stem cells derived from rat fibroblasts (RF) will nowbe discussed. Study of the potential regenerative features of ³⁹K, ⁶⁴Znand ²⁴Mg components on the rat stem cells (fibroblasts) in the area ofdamage to the monolayer has shown that the investigated components didnot change any indicators of the restorative properties of the rat stemcells (RF) after the damage of the cell monolayer. The effect of allcomponents consisted in 100% maintenance their restorative features, andno changes in the migration rate within 72 hours were detected. Therestorative abilities of both processed and unprocessed (control) cellswere the same during this time.

Concentration differences among the components within the experimentwere within fairly narrow limits. Treatment of RF stem cells with lightisotope ²⁴Mg at doses of 2 and 4 mg/ml showed better result at the doseof 2 mg/ml. The cell monolayer was fully restored in 72 hours after theuse of this dose while the dose of 4 mg/ml produced restoration time 10hours longer and amounted to 82 hours.

Treatment of stem cells of RF line with ³⁹K isotopes showed a similartrend. Doses of 1 and 2 mg/ml were tested. Culturing of cells in thepresence of ³⁹K at a dose of 2 mg/ml resulted in complete restoration ofthe cell monolayer in 96 hours of the control time wherein FIG. 123Cshows the result of the action of ³⁹K at a dose of 2 mg/ml after 72hours with an incomplete closure of the edges of the scratch during thistime. At the dose of 1 mg/ml restoration of the monolayer took 72 hoursas shown in FIG. 123D.

Cumulative assessment of the effects of light isotopes ³⁹K, ⁶⁴Zn and²⁴Mg on the migration activity of tumor cells of A-549 and RF cell linesshowed the following. A possibility of suppression of the migrationactivity of A-549 tumor cells of human non-small cells lung cancer using³⁹K, ⁶⁴Zn and ²⁴Mg components has been discovered. The experiment withtumor cells showed a direct dose-dependent effect for ⁶⁴Zn (enhancementof the component effect by increasing its dose), and the effect of ³⁹Kwas greater when it was used at a dose of 2 mg/ml compared to that of 3mg/ml. The concentration characteristic of ²⁴Mg was expressed by abetter effect of the dose of 4 mg/ml compared to that of 3 mg/ml.

⁶⁴Zn isotopes showed the best efficiency both in the experiment with RFstem cells and in the experiment with the tumor cell line. RF stem cellstreated with ⁶⁴Zn at a dose of 25 mcg/ml as well as the control group ofcells (no treatment) completely restored the damaged monolayer within 72hours after the start of the experiment. The time of restoration of thecontrol monolayer of the A-549 tumor cells (not exposed to the action ofisotopes) was 24 hours. The best effect of ⁶⁴Zn at the doses of 20 and30 mcg/ml was in the highest suppression degree of the migrationactivity of the A-549 tumor cell line in the experiment within 72 hours.This is indicative of an expressed selective effect of ⁶⁴Zn specificallyon tumor cells which consists in substantial suppression of theirmigration activity which in its turn is indicative of reduction of theircancerous and metastatic potential. In concurrence with this, anundisturbed restorative potential of the so-called “wound surface” offibroblasts in the experiment with RF stem cells is indicative ofabsence of any changes in their restorative characteristics.

The effect of the Doxorubicin has also been tested on stem and tumorcells. As a result of the effect on the tumor cells, they not onlyfailed to lose their migration and metastatic activity but in oppositeit increased as compared with the control group of cells. Itcharacterizes Doxorubicin as an agent that increases a malignantpotential of a cell and thus the risk of metastases. The effect of thisdrug on the RF stem cells was expressed by a significant decrease intheir overall viability and loss of their restorative function. Thecumulative observational data shows the absence of any inhibiting effecton the metastatic potential of the cells of human non-small cell lungcancer with accompanying significant impairment of the functions of RFstem cells.

Morphological characteristics of the 549-A tumor cells after theirexposure to Doxorubicin showed changes in the adhesive properties of thecells which were expressed in the change of their shape frompredominantly equiaxial as shown in FIG. 114-A towards prolate andoriented in the direction of the migration front as shown in FIG. 114-B,which suggests a change (decrease) in their adhesive properties andadhesive interactions of the cells both with the base and with eachother.

In the general case these changes are a result of disruption of theformation of focal contacts and are manifested in a worse adhesion ofthe cells to the matrix. This leads to changes in the cell activity andthe nature of their motion. The observed effect of Doxorubicin ischaracterized by changes in the factors that stimulate cellmovement-motogenic cytokines. By binding to specific receptors on thecell surface, these factors usually cause stimulation of the cellmobility and proliferation thus enhancing the malignant potential ofcells.

Analysis of activity of ²⁴Mg component on the A-549 tumor cells in theconcentrations of 3 and 4 mg/ml showed a positive trend towardsinhibition (decrease) of the migration activity of the tumor cells ofhuman non-small cell lung cancer. Concentration of 4 mg/ml showed thebest result during the observation period. The effect of ²⁴Mg on theA-549 cells was directly-proportional to the concentration used.

Positive dynamics of the component effect on the RF stem cells wascharacterized by the less time required for restoration of the monolayerafter reducing the dose from 4 mg/ml (and the time of closing the gap inthe range of 75 to 82 hours) to 3 mg/ml with the restoration time of65-72 hours.

Processing of the RF stem cells with Doxorubicin, a reference drug, at adose of 15 ng/ml showed the following results. The dose used in thisexperiment resulted in the irreversible damage of cells, i.e., the celldeath, as well as in substantial decrease in the rate of migration ofthe RF stem cells as shown in FIGS. 122, 123B. Within 96 hours afteradministration of Doxorubicin, the effect of the drug resulted in thedeath of 95% of cells, and the monolayer still was not restored duringthis time.

Combined effect of the component containing ⁶⁴Zn and Doxorubicin on stemcells from rat fibroblasts will now be discussed. In the study of thecombined effect of the preparations, several changes were made in theprotocol of cell processing: cells (5×104/well) were seeded into wellsof a 12-well plate in 2 repetitions in DMEM culture medium supplementedwith a 10% serum and 40 mcg/ml of gentamicin and incubated in 5% CO₂humidified atmosphere at 37° C. for 24 hours. Then the preparationsunder study—⁶⁴Zn isotope containing material, Doxorubicin as a referencedrug, and a mixture of both components—were added in the appropriatewells in various doses. Then the plate was incubated in a CO₂ incubatorfor another 48 hours.

Next, we used a sampler tip (up to 200 mcl in volume) to damage themonolayer. After that we replaced the medium in each well to removecellular debris and smooth the edges of the scratch. The rate of cellmigration was analyzed using an inverted microscope and the cells werephotographed in the region of damage. The migration activity of cellswas fixed at several time points (in 1, 24, 48 and 72 hours depending onthe test cells) after the monolayer damage. The photographs wereanalyzed and the cell incubation time needed to restore the monolayerwas determined for all experimental groups.

During the study of the drug effect on normal cells (fibroblasts) of theRF line in the region of damage of their monolayer it was found out that⁶⁴Zn component (alone) did not affect the rate of migration of cells ofthe RF cell line: the cells processed with this preparation at a dose of25 mcg/ml, as well as the control cells, completely restored the damagedmonolayer in 72 hours after the start of the experiment as shown in FIG.126-D.

Processing of the RF cells with the reference drug Doxorubicin at a doseof 15 ng/ml or 5 ng/ml resulted in a significant decrease in the rate ofRF cell migration. The cell interval was not restored within 72 hours ofthe control time as shown in FIGS. 126-B, C.

The effect of Doxorubicin was also characterized by the death of 95% ofcells 48 hours following the monolayer damage (96 hours afteradministration of Doxorubicin and 48 hours after the drug withdrawal).RF cell culturing in the presence of combination of ⁶⁴Zn at a dose of 25mcg/ml and Doxorubicin at a dose of 15 ng/ml also resulted in the celldeath within 48 hours after administration of the preparations. The datawith regard to the combined effects of ⁶⁴Zn component and Doxorubicinare similar to those produced by Doxorubicin alone.

Migration activity of normal rat kidney cells (NRK cell line) aftertheir exposure to the action of components containing ³⁹K, ⁶⁴Zn and ²⁴Mgcompared to the effect of Doxorubicin will now be discussed. Analysis ofmigration of cells of the NRK cell line in the region of damage of theirmonolayer after they were processed with the materials containing ³⁹K,⁶⁴Zn and ²⁴Mg and Doxorubicin as a reference drug showed that onlyDoxorubicin significantly affected (by slowing the activity) the rate ofcell migration of the NRK line: cells processed with this drug at a doseof 15 ng/ml restored the damaged monolayer only in 72 hours after thestart of experiment as shown in FIGS. 130-A, B. Treatment of NRK cellswith ³⁹K, ⁶⁴Zn and ²⁴Mg isotopes had no effect on their migrationactivity: in 30 hours after the scratch of the monolayer, as in thecontrol, the NRK cells processed with various doses of the experimentalpreparations restored the damaged monolayer.

Comparison of the data on the cell migration activity obtained on tumor(A-549), normal (NRK) and stem (RF fibroblasts) cells after theirexposure to the action of the preparations under study showed thefollowing. The action of ³⁹K component on the A-549 tumor cell line asshown in FIGS. 114, 115, 116C, D consisted in suppression of theirmigration activity within 72 hours (24 hours in the control), and asimilar pattern was observed on stem RF fibroblasts: ³⁹K did not changenatural restorative properties of cells and they restored the monolayerin 72 hours (with 72 hours in the control). The action of this componenton normal cells (NRK) also caused no changes in time—the control groupof cells and cells processed with K restored their monolayer in 30hours.

The effect of ⁶⁴Zn on tumor, normal and stem cells can be characterizedby the following.

Stem cells (RF), after their exposure to this isotope, fully retainedtheir restorative properties with monolayer restoration time of 72 hourswhich is the same as in control group of cells. Normal cells (NRK),after being processed with the component, as well as the control cells(no processing) fully restored the damaged monolayer in 30 hours. As aresult of effect of ⁶⁴Zn on the A-549 tumor cell line the restorationtime of the cell monolayer increased to 72 hours compared to 24 hours ofrestoration of the control monolayer component with no use of isotope.These data demonstrate a selective effect of ⁶⁴Zn specifically on tumorcells which consists in substantial suppression of their migrationactivity which in its turn is indicative of reduction of theirmetastatic potential. In concurrence with this, an undisturbedrestorative potential of the so-called “wound surface” of fibroblastsand normal cells is indicative of absence of any changes in thebiological characteristics in normal cells after the effect of Zncomponent.

²⁴Mg isotope at a dose of 4 mg/ml also shows the ability to inhibit themetastatic activity of lung cancer A-549. At the same time processing ofthe NRK cells with ²⁴Mg had no effect on their migration activity:within 30 hours after the monolayer violation, as in the control, theNRK cells restored the damaged monolayer maintaining the properties ofnormal cells. The comparative characteristics of the effect ofanticancer drug Doxorubicin on various cell types showed the following.A-549 tumor cells of the human lung cancer not only failed to lose theirmigration and metastatic activity but on the contrary, the degree oftheir malignancy after the action of doxorubicin increased withreduction of the time of closing up of the cell gap.

The effect of Doxorubicin on normal kidney cells (NRK) was characterizedby suppression of their restorative ability—the cells processed withthis drug at a dose of 15 ng/ml restored the damaged monolayer only in72 hours after the start of the experiment (compared to 30 hours for thecontrol group of cells).

Combined effect of the component containing ⁶⁴Zn and Doxorubicin on themigration activity of healthy human skin cells (HaCaT cell line) willnow be discussed. The study of migration activity of human keratinocytesof the HaCaT cell line (healthy skin cells) after the combined effect ofZn component and Doxorubicin showed the following results. Separateeffect of Doxorubicin was expressed by slowing down the rate ofmigration of the healthy skin cells as shown in FIGS. 133-B, C. Afterprocessing the HaCaT cells with this drug in 2 doses, 15 ng/ml and 5ng/ml, inhibition of the cell migration rate was observed. The combinedaction of ⁶⁴Zn component and Doxorubicin showed a possibility ofrestoration of the cell monolayer within 36 hours—time required torestore in control group of cells. Thus one more experimentalconfirmation of a positive action of light isotope ⁶⁴Zn was received onthe HaCaT cell line of human keratinocytes—a possibility of reducingnegative effect of Doxorubicin on the regenerative function of healthycells.

Using ⁶⁴Zn without Doxorubicin at a dose of 25 mcg/ml made it possibleto carry out an assessment of the time of the monolayer healing ascompared with the control group of cells. As the experiment showed asshown in FIGS. 133-A, D a separate action of Zn on the HaCaT cell lineof human keratinocytes did not affect the time of monolayer healing. Itwas equal to the time of the control cells monolayer healing—36 hours.

Combined effect of the component containing ⁶⁴Zn and Doxorubicin on themigration activity of human epidermoid carcinoma cells (A-431 cell line)will now be discussed. Analysis of the migration activity of the tumorcells of A-431 cell line as shown in FIGS. 134, 135, 136, 137, 138,which were exposed to the combined action of ⁶⁴Zn and Doxorubicin showedincrease in time for repair of the tumor cells monolayer after the useof light isotope zinc (from 48 hours of control cells repair to 72 hourswith ⁶⁴Zn) and reduction of the time to close up a cellular gap from 48to 45 hours after the use of Doxorubicin. The combined effect of ⁶⁴Znand Doxorubicin also did not differ in character from the resultsobtained earlier. Light isotope neutralized the negative effects ofDoxorubicin completely which resulted in healing the cell monolayer in72 hours and not differ from the result obtained after the action of⁶⁴Zn isotope only. Reduction of the time for the cell monolayer repairfrom 48 to 45 hours after the use of Doxorubicin was detected at a doseof 0.1 mcg/ml as shown in FIG. 137 Administration of a smaller dose of0.02 mg/ml showed the result like in control group—48 hours.

Combined effect of the component containing ⁶⁴Zn and Doxorubicin on themigration activity of human melanoma tumor cells (MM-4 cell line) willnow be discussed. Analysis of a series of photographs, as shown in FIGS.139, 140, 141, 142, 143, illustrating the process of migration of tumorcells of the MM-4 cell line in the region of damage of their monolayershowed that the combined effect of ⁶⁴Zn component, as shown in FIGS.139, 140, 141D, and Doxorubicin at a dose of 0.1 mcg/ml, as shown inFIGS. 139, 140, 141B, suppress the migration rate of MM-4 melanomacells.

The closing up time for the control group of cells not exposed to theaction of the components was 48 hours. Culturing was carried out usingpreparations in the following concentrations. Light isotopic ⁶⁴Zn wastested separately at a dose of 15 mcg/ml and in a combination withDoxorubicin (15 mg/ml of ⁶⁴Zn+0.1 mcg/ml of Doxorubicin and 15 mcg/ml of⁶⁴Zn+0.01 mcg/ml of Doxorubicin), and a separate effect of Doxorubicinwas tested in 2 concentrations: 0.1 mcg/ml and 0, 01 mcg/ml.

When culturing cells in the presence of both preparations—⁶⁴Zncomponent+Doxorubicin at a dose of 0.01 mcg/ml, as shown in FIGS. 139,140, 141E, and ⁶⁴Zn component at a dose of 15 mcg/ml+Doxorubicin at adose of 0.01 mcg/ml, as shown in FIGS. 139, 140, 141F, a significantsuppression of migration activity of the MM-4 cells was recorded. Thetotal time of the monolayer restoration after the combined use of ⁶⁴Zncomponent and Doxorubicin (in 2 concentrations) was 70 hours with 48hours for the control group of cells.

Processing of cells of the MM-4 cell line with Doxorubicin alone showedno statistically significant change in the time of closing of thecontrol monolayer. Observation of the effects of this component at 2concentrations showed that the period of closing of the gap was 48hours, which coincides with the time for the control cell monolayer, asshown in FIGS. 141B, C. Thus suppression of the migration activity oftumor cells was recorded after a separate use of light isotope ⁶⁴Zn onthe MM-4 melanoma cell line (the time of the gap closing amounted to 72hours vs. 48 hours in the control group). In combination withDoxorubicin the time of the gap closing was equal to 70 hours. Thecombined use of ⁶⁴Zn+Doxorubicin demonstrated effect of neutralizationof the negative effects of Doxorubicin. The cell gap was restored in 70hours versus 72 hours after the use of ⁶⁴Zn without Doxorubicin.

Analysis of cellular activity in the Scratch Assay Migrations experimentin which the group of ³⁹K, ⁶⁴Zn and ²⁴Mg isotopes was used showed apossibility of a delay in the time of restoration of the damagedmonolayer of tumor cells and hence the suppression of their migrationactivity and metastatic potential. A comparative assessment of the timeis characterized by 72 hours required for closing the cell gap when ³⁹K,⁶⁴Zn and ²⁴Mg isotopes are used separately (on 3 tumor cell lines—A-549,A-431 and MM-4) compared to 24 hours needed to restore the monolayerwithout the use of the components on the A-549 cell line and 48 hours onthe A-431 and MM-4 cell lines.

Assessment of the effects of ³⁹K, ⁶⁴Zn and ²⁴Mg components on normal(NRK), stem (RF) and skin (normal human keratinocytes HaCaT) cellsshowed their ability to maintain restorative and regenerating propertiesof stem cells. The time of restoration of the cell monolayer after theuse of ³⁹K, ⁶⁴Zn and ²⁴Mg components did not differ from the time forcontrol groups of all cell lines (NRK, RF, HaCaT). A positive effect of³⁹K, ⁶⁴Zn and ²⁴Mg on normal stem cells expressed in the absence of anycontact inhibition effect during the formation of cell monolayer on theNRK, FC, HaCaT cell lines. Activity of the control and experimentalgroups of cells showed the same results with respect to the time of thecell monolayer restoration which is indicative of the absence of anydisturbances of the cell growth mechanisms in the experimental groupwhere ³⁹K, ⁶⁴Zn and ²⁴Mg were used and of the possibility of maintainingregenerating properties of cells in the presence of light isotopes.

Changes in the morphological characteristics of tumor cells of the A-549cell line after their exposure to the action of Doxorubicin weredetected. They were expressed in the change of their shape frompredominantly equiaxial, as shown in FIG. 114-A, towards prolate andoriented in the direction of the migration front, as shown in FIG.114-B, which suggests a decrease in their adhesive properties andadhesive interactions of the cells both with the base and with eachother. Said changes are due to disruption of the formation of focalcontacts and are manifested in a worsening adhesion of the cells to thematrix. This leads to changes in the cells activity and the speed oftheir movement. The observed effect of Doxorubicin is characterized bychanges in the factors that stimulate cell movement-motogenic cytokines.By binding to specific receptors on the cell surface, these factorsusually cause stimulation of the cell mobility and proliferation thusenhancing the malignant potential of cells. The effects of lightisotopes ³⁹K, ⁶⁴Zn and ²⁴Mg on the A-549 cells after 72 hours is adecrease in the number of unequiaxial cells as shown in FIGS. 116-C, E,G.

The effect of anti-cancer drug Doxorubicin on the migration of theA-549, A-431 and MM-4 tumor cells was characterized by increasedcellular activity and reduced time of the cell gap closing as comparedto the control groups of cells. It means that Doxorubicin increasesmetastatic potential of tumor cells. Treatment of stem cells withDoxorubicin at a dose of 15 ng/ml resulted in the cell death. The effectof Doxorubicin on the RF cell line resulted in the death of 95% of cellswithin 96 hours. The effect of this component on the RF stem cells was asignificant reduction in their overall viability and loss of theirnatural regenerative function.

Action of Doxorubicin on tumor cells is characterized by increased cellactivity and, as a result, an increase in their metastatic potentialwhich is manifested in accelerated (as compared to the control group ofcells) closing of the cell gap. The action of ⁶⁴Zn component, on thecontrary, leads to reduction of the tumor and metastatic activity ofcells which was manifested in the increase of the time of the cell gapclosing as compared to the control group cells.

Comparative assessment of the effects of the pair of ⁶⁴Zn-Doxorubicinshowed that the combined use of these components on tumor cell linesalso reduces migration and tumor cell activity of A-549, A-431 and MM-4cell lines. This makes it possible to use ⁶⁴Zn containing materials bothas an independent anti-tumor component, and in the complex schemes ofcombined therapy aimed to protect organs and tissues from the toxiceffects of anti-cancer drugs and their decomposition products(metabolites).

Summarizing the study of the combined effects of ⁶⁴Zn and Doxorubicinthe following conclusions can be made: ⁶⁴Zn isotopes do not affect therate of migration of normal stem cells which confirms the absence of anynegative effects on normal cells. Anti-migration and, as a consequence,anti-metastatic effect of ⁶⁴Zn on tumor cells has been observed, whichis important, since the degree of migration mobility of malignant cellsis the main characteristic of their metastatic and malignant potential.⁶⁴Zn component has an ability to suppress adverse effects of anti-canceragents, namely inhibit increased migration of tumor cells after theaction of antineoplastic Doxorubicin.

Alluding to the above, the aforementioned results prove that it isbeneficial to use ⁶⁴Zn isotopes in combination with any standardantineoplastic therapy in order to improve its efficiency and reduce theharmful effects of anti-tumor agents. Preparation of samples will now bediscussed. Experimental samples obtained by surgery or filtrationdepends on the objects of investigation (blood, lymph etc.). Saidsamples having a mass of about 1 gram were quick-frozen by immersion inliquid nitrogen. To reach high rate s of freezing small portions ofsamples have been used. In ultrafast cooling at a rate of about onehundred degrees in 1 sec., water was transformed into amorphous icewithout volumetric expansion.

After that, the amorphous ice was removed from the sample by means ofvacuum freeze drying at a low temperature. The ice evaporates and themineral particles and organic components of the sample remain in thesame position in which they were in the initial wet sample, and thus thestructure and chemical composition of the sample is preserved. To reducethe drying time of the sample during sublimation, dry nitrogen was fedautomatically to the vacuum chamber. This technique increases thermalconductivity of the sample and therefore the convection heat supplyaccelerates the process of sublimation. The gas feed rate to the vacuumchamber did not exceed 0.1 l/min. Samples drying time under theseconditions was 10 hours.

At the final stage of preparation of the samples an additional dryingunder the high vacuum was done. At almost complete (99%) dehydrationunder the high vacuum, samples were heated to a temperature of 35-40° C.and then have been aged isothermally for about 1 hour under theseconditions. Method of secondary ion mass spectrometry with Cameca IMS-4Fmass spectrometer was used. Mass spectrometry in the biological sampleswas performed based on the m/z ratio analysis in the pulse counting modeas well as using results displayed as a mass-spectrum as shown in FIG.145 or as a profilogram that characterizes distribution of the detectedmasses along the sample plane as shown in FIG. 147 and in depth as shownin FIG. 146.

The sample prepared for the experiment was placed in a sample holder.After evacuating the system, the probe was calibrated and its mode wasstabilized. To prevent the destructive action of the ion beam on thesample and to reduce the amount of static electricity caused byionization, the analysis was carried out at very low rates of the samplesputtering (less than 104 of the monolayer per second).

A subsequent analysis of the masses obtained in result of the ion beambombardment of the sample surface was based on the interaction ofsecondary ions ejected from the sample surface with the electric andmagnetic fields of the detectors. Utilization of a double-focusingspectrometer with combination of electric and magnetic fields controlsmade it possible to maximize the sensitivity of the instrument. For suchmultistage magnetic spectrometers a background signal resulting from theresiduals of the main peaks of the matrix material (wall scattering, onthe gas atoms, etc.) can be reduced to a level of less than 10-9 for thegeneral background and only 10-6 for all masses close to the main peak.

To minimize the amount of gases adsorbed on the sample surface (H₂, N₂,O₂, H₂O, CO₂ and CO) the measurements were performed in an ultrahighvacuum free of hydrocarbons using cryogenic and getter pumping near thesample. To reduce the formation of positive charge on the surface of thetest sample due to electron ionization, the latter was irradiated withelectrons emitted by a thermal cathode located nearby. In addition, anelectro-conductive additive to the sample increased samplesconductivity. Sample dried by sublimation was placed on a metal meshwith a mesh size of 25×25 mm using a pressing technique with theaddition of electro-conductive highly dispersed carbon black. Thequantitative content of the electro-conductive additive was 0.1% of thesample weight to drain off accumulated charge on the metal mesh.

For the measurement, several methods were applied using Cameca IMS-4Finstrument: a) mass spectrometry, b) the method of direct imaging ofisotope distribution on the sample surface, c) the method ofprofilograms, which made it possible to assess a degree of homogeneityof the isotope distribution in the depth of the sample as shown inFigures

A vertical line in the middle part corresponds to the naturaldistribution for each of the detected isotopes. Horizontal yellow andgreen diagrams characterize deviations from the natural content for eachof the said isotopes. Therefore the left side of the graph (with shadeddiagram strips) corresponds to an increase in the concentration ofisotopes and is represented mainly by heavy isotopes, while the rightside (with unshaded diagram strips) on the contrary-a decrease in thepercentage of isotopes in relation to the natural content primarily dueto light isotopes.

FIG. 148 illustrates the character of distribution of isotopes in the“young” and “old” tissues. We also analyzed the isotopic ratios inpathological (cancer) and healthy tissues of the following chemicalelements: magnesium, silicon, sulfur, chlorine, potassium, calcium,chromium, iron, nickel, copper, zinc, bromine, rubidium, molybdenum andsilver.

The following types of biological tissues were studied, as shown inFIGS. 155, 156, 157, 158 are biological tissues containing canceroustumor cells (sample 20 and sample 12) where sample 20 had metastases,normal biological tissues (sample 12 and sample 18). Within each pair ofsamples (tumor and normal tissue were divided into the following pairs:12-14 and 18-20), the pairs of samples belonged to the same biologicalorganism. Isotope distribution was studied on the following pairs ofsamples (see Table 1). Samples 12 and 14, where sample 12 was taken fromthe central area of the tumor and sample 14 from normal tissue. 2).Samples 18 and 20, where sample 18 was taken from normal tissue andsample 20 from tissue affected by the tumor. Distribution of isotopesdetected in samples 12 and 14 is shown in FIG. 8 and in samples 18 and20 in as shown in FIGS. 159, 160.

Comparative assessment of the experimental results on samples 12 and 14is shown in as shown in FIGS. 157, 158 and on samples 18 and 20 in asshown in FIGS. 159, 160. Analysis of the obtained data evidences thefollowing pattern of fractionation of the isotopic composition in thetissues affected by the tumor. Tissue samples 20 and 12 characterizingtumor tissues had the following factors of similarity and differences.The similarity consisted in their belonging to the tumor tissues and thedifference was in the presence of metastases of poorly differentiatedtransitional cell carcinoma in sample 20, which characterized thisbiological sample as an object with a more pronounced degree of damageby cancer.

When analyzing the results of obtained on samples 12 and 14, it can befound that of 20 isotopes detected in these samples represented bymagnesium, silicon, potassium, iron, copper, zinc, bromine, rubidium,and silver, the major part, in the amount of 11 isotopes, ischaracterized as heavy isotopes primarily concentrated in the tissueaffected by the tumor (in the “cancer tissue” column in FIG. 157, theseisotopes are highlighted in red).

This is in correspondence with the data as shown in FIGS. 155, 157, 158,wherein the content of heavy isotopes in the normal and cancer tissuesis shown in red, and the analysis of these results reveals that heavyisotopes is observed in the normal tissue as well. However, the generaltrend is accumulation of heavy isotopes in the tissue affected by thetumor.

Now let us consider the results obtained on samples 18 and 20. Sample 18was represented by healthy tissue and sample 20 was a metastasis ofpoorly differentiated transitional cell carcinoma with extensivenecrosis. It should be noted that compared with samples 12 and 14 thesesamples contained a much larger number of both the elements andisotopes. This can be explained by the man-caused factors of existenceof a biological organism as well as by certain selective conditions ofaccumulation of some elements and their isotopes in a pathologicaltissue. We have determined that a pair of samples 18 and 20 containednew elements, such as molybdenum, nickel, chromium, chlorine and sulfur,and the list of isotopes found in samples 12 and 14 was considerablyexpanded and included such isotopes as Zn-68, Zn-64, Fe-57.

If we describe the results obtained on samples 18 and 20 statistically,the following can be concluded. Of the 30 isotopes identified in thispair of samples (which are represented by 11 elements such as magnesium,silicon, sulfur, chlorine, potassium, chromium, iron, nickel, zinc,rubidium, molybdenum) 15 isotopes can be characterized as heavy.According to FIGS. 159, 160, which reflect the results of examination ofsamples 18 and 20, one can see a predominant increase in the number ofheavy isotopes in the tumor tissue affected by metastases as compared totheir content in normal tissue. Quantitative assessment of the nature ofdistribution of light isotopes is bidirectional and consists in thefollowing. A slight increase in the content of light isotopes of suchelements as magnesium, sulfur, chlorine, nickel and molybdenum in thehealthy tissue relative to their natural distribution (shown in blue)was observed.

As it was noted earlier (in the examination of young and old tissues aswell as of samples 12 and 14), an increase in the concentration of heavyisotopes is accompanied by a decrease in the concentration of lightisotopes. This is also true for samples 18 and 20. It is important tonote a few more facts that characterize the correlation between theconcentration of heavy isotopes and a degree of disease of biologicaltissue. Sample 20 was taken from metastases of poorly differentiatedtransitional cell carcinoma with extensive necrosis and sample 12—ofrenal cell carcinoma. From a medical point of view, a metastasis is arecurrence of cancer and it is more dangerous and serious complicationfor the life of a patient than the primary tumor. The poorlydifferentiated cancers have the most adverse outcomes. This happensbecause the tumor overcomes protective barriers and cancer cells get inthe lymph and blood stream.

Quantitative assessment of the isotopes detected in samples 12 and 20indicates an increase in their number (in sample 20) along with anincrease in the degree cancer aggressiveness. The range of deviation ofisotope concentrations from the one in natural isotope distribution ismuch higher in sample 20. The maximum deviation from the natural contentfound on silicon isotopes in sample 20 with an increase in Si29 by11.6%, while the content of Si28 isotope reduced by 20.6%. Such adifference is expressed less clearly in the pair of samples 12 and 14.The largest increase here was observed in the content of Rb87 isotopewhich amounted to +8.35% in the tissue affected by tumor, while thelargest decrease in the isotopic concentration was found in Mg24 isotopewhich was −8.1%.

This trend is observed on other elements as well. However, a shift inthe distribution towards heavy isotopes is considerably stronger in thepair of samples 18 and 20. Biological tissue affected by cancer cellsand metastases has a higher concentration of heavy isotopes while thebordering normal tissue has a lower concentration of light isotopes.Results of mass spectroscopic study of isotopic composition of samplesof fungus and cortex. Analysis of the content of trace quantities ofisotopes requires prior sample preparation as elements in most objectsare in a bound state. They form quite strong organic complexes thatprevent accurate and reproducible determination of their content.Therefore, prior to any analysis it is necessary to destroy the organicportion of the sample. Preparation of the samples of fungus and cortexfor the analysis was carried out using the dry ashing method. The dryashing method involves sequential heating of a substance to the ashingtemperature in oxygen in a closed system.

Dry ashing of the samples of fungus and cortex was performed in aceramic crucible placed in a muffle furnace. For the destruction of theorganic bond of substances the same quantities of the samples weighedfor the analysis (6 grams each) were used. The samples in crucibles wereplaced in a muffle furnace without protective atmosphere (i.e. in air)at the temperature of 150° C. At this temperature, the samples wereheated for one hour and then they were dried step by step by raising thetemperature of the furnace by 50° C. every hour. The temperature of thesample was raised in such a mode up to 350° C. (for 4 hours), whereuponit was raised to 360° C. The samples were then held at this temperaturefor 20 minutes.

Then crucibles with ash were extracted from the furnace and were cooledunder the atmospheric conditions to the room temperature. With the sameinitial masses of fungus and cortex, after completion of the dry ashingprocedure the fungus weighed 112 mg while the cortex weighed only 32 mg.The solid residues of the samples of fungus and cortex were pressed in anickel metal mesh with a mesh size of 50×50 microns. The metal meshhelped to keep the sample material fixed and additionally served as thecurrent collector reducing the amount of static electricity caused byionization by the ion beam. Information on the isotopic composition ofthe samples was obtained from the surface area 200×200 mm in size.

Analysis of the samples of fungus and cortex was performed with respectto the following detected elements: magnesium, silicon, potassium,calcium, titanium, iron, copper, zinc, rubidium, strontium. The totalnumber of isotopes found in the samples was 36 isotopes in the 10detected elements. Quantitative assessment of each isotope was obtainedbased on the primary analysis of the experimental data characterizingthe relationship of mass and impulse response for each of the isotopes.

FIG. 161 show the results of quantitative assessment of the content ofisotopes in fungus and birch cortex, and the diagram in FIGS. 161, 162makes it possible to visually compare the quantitative characteristicsof isotopic distribution in fungus and the birch cortex and rings. Thefollowing conclusions were made based on the results of our study: Insamples from the interior of the tree (birch ring), the naturaldistribution of isotopes was observed. In the samples prepared from thefungus, the isotopic distribution shifted toward the light isotopes. Theconcentration of light isotopes was considerably higher and theconcentration of heavy isotopes was considerably lower than in thenatural distribution.

In the samples prepared from the cortex, surrounding the fungus theisotopic distribution shifted towards an increase in a portion of heavyisotopes and a reduction in the concentration of light isotopes. Itshould be noted, that the fungus itself is mechanically strong while thecortex around it is in fact quasi-dead tissue with no mechanicalstrength.

In accordance to modern science, one would never expect any deviationsfrom natural distribution of isotopes for a given element. It does notmatter, which object of nature that this element belongs to, and whereit is in our universe. For example, potassium is a mixture of twoisotopes K-39 (93.3%), K-41 (6.7%) and long-lived radioisotope of K-40(0.012%).

Substitution of one isotope on another may result in the isotopeeffect-kinetic or magnetic isotope effect, isotopic shift or smallvariation in the temperature of superconductive transition. Isotopeeffect should not cause any dramatic changes in chemical properties orstructure of materials.

The aforementioned is correct for non-live matter but not for microbesand not for animals and not even for viruses or plants. The results andexperimental conditions of detailed study of isotope distributionpeculiarities in healthy and pathology affected biological tissues, aswell as in “young” and “old” tissues at present stage were obtained onthe level of tissue and are not protein or amino acid specific. Neverthe less said results are very important and allow for new ways ofdiagnostics, treatment and prevention of irreversible pathologiesincluding cancer and ageing.

Although samples represented different types of cancer or “young” and“old” tissues, the conclusions made have much broader significance, asmany pathologies can be discussed in terms of extreme local ageingeffects. Notion of immune system should be used not only to describebody's defense against microbes, viruses and other invaders, but alsothe ability to repair mutation-threatening damage to the most importantmacromolecules. This function is crucial for irreversible diseaseprevention and as proved by our data requires uninterrupted critical“spare parts” supply. Said “spare parts” being set of lightest isotopesof vitally important metals. Mass-spectrometry data shows that inhealthy tissue of not too young and too old person there is so-callednatural distribution of isotopes in complete correspondence with alltextbooks. In the tissue from the body of 77 years old, there is a shiftin isotope distribution to the side of heavy isotopes.

Concentration of light isotopes in a number of elements is significantlydecreased and concentration of heavy isotopes is correspondentlyincreased. Shift of isotope distribution in favor of heavy isotopes wasalso observed in samples affected by cancer tumor. There is correlationbetween severity of disease and concentration of heavy isotopes in thetissue. In the sample taken from malignant tumor with well-expressedmetastases, concentration of heavy isotopes was much higher and forlight isotopes-much lower than in the tissue of malignant tumor withoutmetastases.

In contrast to the “old” and cancer affected samples, in “young” tissuethe shift of isotope distribution in the favor of light isotopes wasdetected with increased concentration of light isotopes and significantdecrease in concentration of heavy ones. The difference inconcentrations of heavy and light isotopes in healthy and cancer tissuesof the same patient depends on progress of the disease and can be higherthan tenfold. It is important to notice that as much as light isotopesare spare parts to fix broken chemical bonds back to normal, the heavyisotopes are also spare parts without which dangerous damage to thechemical bonds cannot hold on in time or stabilized and eventually isrepaired. It is logical to expect that in right amount light isotopesshould be able to rejuvenate biological tissue and transform cellsaffected by pathology back to normal state. At the same time, one canconsider heavy isotopes of the same elements as an efficient andmultipotent pathogen or kind of a poison.

At certain concentration, they can cause most of the broken chemicalbond to become a source of irreversible pathology. Concentration oflight isotopes is also a key to successful treatment of variousillnesses. To achieve positive result the amount of “cure” should bemuch higher than amount of“poison”. A lot depends on the damageinflicted by disease. That is why it is inappropriate to speak of oneuniversal dose of “cure” for any disease at any stage. The target is toprevent access of heavy isotopes and provide right amount of lightisotopes supply during the treatment time. Otherwise, results will byinconclusive and irreproducible creating uncertainty and confusion. Letus take two examples to demonstrate what happens: one from biotechnologyand another from pharmaceutics/folks medicine. First, stem cellstreatment. It is proclaimed as possible universal cure and sometime itreally helps to some patients with some illnesses. To a few and notalways, and no explanation why.

Let us put aside problems related to the reaction of immune system onthe alien stem cells infusion. Then stem cells should be able to helphealthy organism to become stronger due to the regenerative ability andtheoretical possibility to differentiate and substitute damaged cells ofvarious organs. It is necessary to add—given there is sufficient supplyof key light isotopes to the tissue or organ under discussion.Otherwise, the lack of light isotopes and excess of heavy ones may causetransformation of stem cells into cancer cells. Stem cells aredefinitely “young”. Therefore, their positive effect can be attributedto the fact that they contain higher that natural concentration of lightisotopes.

Here we speak of ratio between light and heavy isotopes. Absolute amountof light isotopes in stem cells is very low. In general, stem cells canbe considered as source and carriers of very low quantities of lightisotopes. It is better than nothing and sometime even helpful, but it isdifficult to predict when. Second example is birch treemushrooms-“chaga”. It is believed to be a “virtual cocktail ofantioxidants and phytonutrients” able to bust immune system, reduceinflammation and eliminate cancer. It was used for many centuries asfolk medicine with universal properties. Nobel Prize winner AlexanderSolzhenitsyn believed he was cured from cancer with chaga and describedthe story in his famous book “The Cancer Ward”. Yet, there is no soliddata or statistics to support the legend. They say taking chaga tea overa long period is extremely beneficial. It is probably true. At the sametime, nobody dares even to speculate on probability to cure any disease,let alone cancer, with chaga in less than a lifetime.

To figure out the nature of legendary claims on chaga properties we haveconducted mass-spectrometry study of samples prepared from chagamushroom, surrounding bark and birch rings (inner volume of the tree).Results should help to understand real reasons of biological activityfor not only chaga, but other folk recipes as well. Chaga mushroomappeared to have a really unique and unheard feature-isotopeselectivity. Like in the young human tissue, the isotope distribution isdifferent from the natural one with much higher concentration of lightisotopes and lower concentration of heavy isotopes in chaga samples,reverse picture (like in cancer affected tissues) in surrounding barkand natural distribution deep inside birch rings/trunk. They believethat birch tree host gradually dies of due to the mushroom consuming allnutrients from the bark and trunk. At the same time, nothing ispreventing additional supply from the root system.

The real problem is the constant excess of heavy isotopes over lightones in the bark and trunk around the mushroom. That is what killshealthy tissue. Chaga contains up to 30% higher concentration of lightisotopes of vitally important metals like K, Zn, Mg and Rb. There is asignificant shift in isotope distribution but heavy isotopes are stillpresent although in a smaller amounts. We have also discovered thatlight isotopes spread not equally over the samples. It means that thereare areas in mushroom cross-section with no light isotopes at all.Hence, seemingly random biological activity of chaga extracts can beexplain by Competition between effects of higher than normal amount oflight isotopes and less than normal but still significant presence ofheavy isotopes.

Hundreds to thousands times smaller quantities of light isotopescompared to the daily intake of vital elements. Non-homogeneousdistribution of chemical elements in the bulk of mushroom. It means thatquite often extracts are made from chaga's parts that do not contain anyuseful elements at all. Chaga's extract consumer should be lucky enoughto have multiple random events to coincide to get theoretically possibletherapeutic effect.

In Vivo Experiments with Ascites

The term “malignant ascites” is understood as a pathologicalaccumulation of fluid in the peritoneal or pleural cavity, whichdevelops as a result of tumor damage to the peritoneum or lungs.Malignant ascites can be caused by a variety of primary tumors, such as,for example, breast cancer, ovarian cancer or gastrointestinalcarcinomas.

The present inventors have unexpectedly found that the agent comprisinga light zinc isotope, ⁶⁴Zn in particular, as an aspartate orasparaginate, as well as the method which comprises administration ofsuch agent containing ⁶⁴Zn as an aspartate or asparaginate at doses from350 μg ⁶⁴Zn_(e) to 461 μg ⁶⁴Zn_(e) via intraperitoneal injection,suppress the development of malignant ascites. The intraperitonealinjection of this agent produces a pronounced inhibitory effect on thedevelopment of cancers causing ascites, mouse leukemia and mouse breastcancer (Ehrlich ascites carcinoma) in particular, and improves survivalin the studies in model mouse systems.

In the experiments on mice, a high antitumor activity of the ⁶⁴Zn_(e)active ingredient was demonstrated with its direct effect on tumorcells, for example, in ascitic fluid.

Light isotope-enriched elements as enumerated above, including ⁶⁴Zn_(e),can be formulated in a variety of dosage forms depending on the objectof use, in particular as solutions for injections, ointments, and thelike. In a preferred embodiment of this invention, the composition forsuppression of malignant ascites further comprises agents that promotepenetration of the active substance into cells, for example DMSO.According to one preferred embodiment of the method for suppression ofthe development of the malignant ascites, the composition of theinvention, such as one comprising ⁶⁴Zn_(e), is administeredintraperitoneally and/or intravenously. It is preferred that theadministration be intraperitoneal.

The studies conducted in mice suggest that the antitumor effect of the⁶⁴Zn_(e) compound is probably due not to the direct cytotoxic effect ofthe drug on tumor cells but indirect one, possibly through theinhibition of proliferation and the activation of nonspecific immunity,as the percentage of dead tumor cells in ascites which were exposed tothe action of the agent according to the invention does not exceed 5%.This is also evidenced by a complete absence of acute toxicity for micewith complete inhibition of tumor growth.

The main advantage of the proposed method is that its use does not causetoxic effects, as is characteristic of most methods based on the use ofknown cytostatic agents, and, due to the important physiological role ofthe substances used in the method, it, in addition to antitumoractivity, provides a number of additional advantages, associated withthe optimization of the catalytic, structural and regulatory functionsof these elements in the body. It should also be noted that the activityof the ⁶⁴Zn_(e) compound for suppressing the development of malignantascites is not inferior to that of known cytostatics.

An aspect of the present invention is described more fully hereinafterby reference to the following examples, which are presented by way ofillustration only and should not to be construed to in any way limit thescope of the present invention.

A pilot study to evaluate the efficacy of the light isotope compositionsand method of using them to suppress the development and treatment ofmalignant ascites in humans has also been conducted.

EXAMPLES Example 6. Zinc (Zn-64) and Aspartic Acid Complex ProductionProcess Preparation Process of ⁶⁴Zn Aspartate

⁶⁴Zn aspartate (racemic) having the following formula (in which “⁶⁴Zn²⁺”refers, in this one instance, to Zn²⁺ enriched for 6Zn) was prepared inthe experiment.

At the first stage, zinc oxide enriched for ⁶⁴Zn was prepared using⁶⁴Zn_(e) sulfate as the starting compound.

⁶⁴Zn_(e)SO₄+2NaHCO₃→⁶⁴Zn_(e)O+Na₂SO₄+2CO₂+H₂O

For this purpose, ⁶⁴Zn_(e) sulfate (zinc was at least 99.9% ⁶⁴Zn,although ⁶⁴Zn_(e) of lower purity may be effective) in an amount of 0.01mole) was dissolved in 150 ml of water (T=50-70° C.) wherein 1.68 g(0.02 mole) of sodium bicarbonate was added in small portions, toprevent severe foaming, with constant stirring in a magnetic stirrer.After completion of foaming the solution was stirred for another 30minutes and then left for 1 hour until a white precipitate was formed.During this process, the temperature was maintained at about 60° C. toprevent crystallization of sodium sulfate. The solution with theprecipitate, which precipitate was ⁶⁴Zn_(e)O, was then filtered withoutcooling. The resulting precipitate—⁶⁴Zn_(e)O—was washed with warmdemineralized water (T=40-50° C.) and dried to constant weight in adesiccator over the dehydrating agent phosphorus pentoxide.

After that, 425 ml of demineralized water was poured into a 1 literflask and heated under reflux to 80° C. 1.33 g (0.01 mole) of asparticacid was dissolved in water with stirring by a magnetic stirrer. Afteraspartic acid was completely dissolved, 0.8 g (0.01 mole) of ⁶⁴Zn_(e)Oobtained at the previous stage was added to the clear solution. Themixture was stirred with heating to 80° C. for 1½-2 hours till completedissolution of ⁶⁴Zn_(e)O. The reaction formula is shown below:

If the precipitate (⁶⁴Zn_(e) oxide) was not dissolved completely, thesolution was filtered and the undissolved ⁶⁴Zn_(e)O was collected anddried to its constant weight to determine the ⁶⁴Zn_(e) complexconcentration in the resulting solution. The solution was transferredinto a volumetric measure and made up to a volume of 425 ml usingdemineralized water. 425 ml of ⁶⁴Zn_(e)-aspartic acid complex containing⁶⁴Zn_(e) in the amount of approximately 0.0015 g ⁶⁴Zn_(e) (1.5 mg⁶⁴Zn_(e))/ml was thus prepared.

In Vivo Experiments

Unless stated clearly otherwise, in the ⁶⁴Zn-enriched zinc aspartate(referred to as “⁶⁴Zn_(e) aspartate”) that is used in examples 7-9, thezinc is 98.73% ⁶⁴Zn. Also, dosage amounts of ⁶⁴Zn_(e) aspartateexpressed in pjg/mouse indicates the amount of ⁶⁴Zn_(e). For example,“75 μg/mouse” of ⁶⁴Zn_(e) aspartate indicates 75 μg of ⁶⁴Zn_(e) permouse. The solutions of ⁶⁴Zn_(e) aspartate consisted of ⁶⁴Zn_(e)aspartate dissolved in deuterium-depleted water.

In order to confirm the efficacy of the method of the present inventionin suppressing the development of malignant ascites, in vivo studieswere performed using a mouse model.

All studies were performed in compliance with the rules of the EuropeanConvention for the Protection of Vertebrate Animals Used forExperimental and Scientific purposes [Commission of the EuropeanCommunities: Council Directive of 18 Dec. 1986 on the Lows, regulatingthe Application of Principles of Good Laboratory Practice and theVerification of Their Applications for Tests on Chemical Substances(87/18/EEC). The Rules Governing Medicinal Products in the EuropeanCommunity.—1991.—V. 1.—P. 145-146].

The research institution followed the requirements of the plan approvedby the Customer and standard operating procedures (SOPs) of thelaboratory. The test material was handled in accordance with standardsafety rules: the personal protective equipment (gloves, mask and whitecoat) was worn, the Material Safety Data Sheet (MSDS) was available tothe staff and the staff was informed of the potential risks andprotective equipment before starting work.

The animals were managed in accordance with the standards set forth inThe Guide for Care and Use of Laboratory Animals (ILAR publication,1996, National Academy Press, 1996). All procedures with experimentalanimals were reviewed for their compliance with the ethical principlesof scientists working with animals and approved by the BioethicsCommission at R. E. Kavetsky IEPOR NAS of Ukraine. The animals were keptin plastic cages with the bedding; the cages were equipped with steellatticed lids and feeding niches. Commercial material, such asecologically clean hardwood chips, was used as the bedding and theroutine checks of the bedding for its compliance with the technicalrequirements were performed. Characteristics: the chips were ofdifferent shapes, 5-20 mm long and 2-1 mm thick, the maximum moisturecontent was 12%, the chips did not contain any harmful impurities suchas heavy metals, pesticides, herbicides or insecticides. A standardpelleted diet for laboratory animals K-12-4 PP “REZON-1”, (Ukraine) wasused. The diet consisted of basic food ingredients, vitamin and mineraladditives. This diet is subject to a regular quality control and thecontent of food components by the manufacturer. The diet samples wereregularly tested for microbiological contamination. The animals weremanaged under controlled environmental conditions (21-26° C. and therelative humidity in the range of 30 to 60%). The temperature andhumidity were constantly monitored. A 12 hour light cycle was maintainedin the rooms where the animals were managed. The animals were identifiedby cage numbers.

Procedure of drug administration: the agent solutions were injectedintraperitoneally in the right inguinal region or intravenously in thelateral tail vein using Micro-Fine Plus microinjection syringe (BectonDickinson). The injection site was pretreated with 96% ethanol.

Clinical observations: daily inspections of all the animals in cageswere carried out in order to determine mortality or any signs ofdeviations in their health status. A thorough examination was performedeach time any abnormalities were detected. All deviations were recorded.The tumor size was measured every 2 days (3 times a week) after thestart of visible ascites/tumor node growth (the 8^(th) day after tumorcell inoculation).

Terminal procedures and pathomorphology: if an animal died in thestudies, the time of its death was determined and recorded, the animalwas weighed and subjected to necropsy (without organ retrieval). Dyinganimals were euthanized, weighed and necropsied.

Descriptive statistics was applied to all the quantitative data: themean value and standard error of the mean were calculated which are,along with the N value, shown in the summary tables. To determine thereliability of the intergroup differences, the data were analyzed byparametric or nonparametric tests depending on the quantitative datadistribution type. The differences were determined at 0.05 significancepoint.

Example 7. Studies Supporting the Efficacy of a Light Isotope-EnrichedCompound and Method Performed in In Vivo Experiments Using a Mouse Modelof Breast Cancer (Ehrlich Ascites Carcinoma)

10 male outbred mice (5 mice per group) at the age of 10 weeks, weighing19-23 g were used in Ehrlich ascites carcinoma (EAC) model. Before theexperiment, all the animals were healthy, with normal behavioralperformance. During the experiment, the animals were kept in plasticcages under natural light illumination, on a standard diet with freeaccess to food and water. An ascites strain of mouse breast cancer (EAC)maintained in outbred mice was used in the experiment. To inoculate thestrain, the tumor cells derived from the ascitic fluid were placed insaline solution. The suspension cellularity was evaluated in thehemocytometer and adjusted to a concentration of 1×10⁶ cells/ml withsaline solution. The tumor was transplanted by injecting 250 l of thetumor cell suspension (0.25×10⁶ cells/mouse) into the abdominal cavityof the animals.

The animals were divided into groups as follows:

-   -   Group No 1—control group, mice with EAC+solvent        (deuterium-depleted water (this water contains deuterium at a        level of 10 ppm));    -   Group No 2−mice with EAC+⁶⁴Zn_(e) aspartate.

The solution comprising ⁶⁴Zn_(e) aspartate was injectedintraperitoneally using a microinjection syringe at a dose of 0.075 mg(75 μg)/mouse every other day (5 times during 10 days) starting from thefirst day after injection in a volume of 0.5 ml/mouse. The dynamics oftumor growth in the experimental animals was observed for 13 daysfollowing the i.p. inoculation of tumor cells (by the volume of ascitesin the peritoneal cavity). On the 13th day after i.p. inoculation oftumor cells, the whole ascites fluid was recovered from the animals andthe total number of live/dead cells in each mouse was determined.Deuterium-depleted water alone was used instead of a solution of⁶⁴Zn_(e) aspartate as control in the experiment.

The dynamics of formation of ascites in the control and experimentalgroups was assessed visually using a 10-point scale (one point was 0.46cm of the mean diameter of the mouse abdomen filled with ascites).

In addition, on the 13^(th) day after intraperitoneal inoculation oftumor cells, all ascetic fluid was removed from the abdomens in allanimals from each group by washing their peritoneal cavities with salinesolution and live and dead tumor cells were counted using thetraditional vital dye trypan blue (HyClon, USA) and a hemocytometer.

The number of cells was determined using the following formula:

X=((a)/80)×10⁶, where X is the number of cells in 1 ml and a is thenumber of cells counted in 80 small squares of the hemocytometer.

The data on the dynamics of accumulation of ascites in the experimentalanimals and lifespan of mice in the in vivo experiment were used toassess anticancer activity of ⁶⁴Zn_(e) aspartate against mouse model ofbreast cancer. The results of the studies are presented in Tables 1-2.

TABLE 1 Ehrlich carcinoma growth dynamics (analysis of changes in thevolumes of ascites in laboratory animals) Group of experimental animalsDay after inoculation of tumor cells 7 9 11 13 Volume of ascites in mice(points) EAC control 2.8 ± 0.6 6.3 ± 0.5 8.1 ± 1.1 9.4 ± 0.6 EAC +⁶⁴Zn_(e) aspartate 0* 0** 0* 0** *p < 0.02, **p < 0.002 as compared withthe control group

The data analysis shows that the agent according to the inventioncontaining ⁶⁴Zn-enriched zinc as aspartate exhibits significantantitumor activity against Ehrlich ascites carcinoma in mice. Thus aseries of 5 i.p. injections of the agent at a dose of 75 μg/mousestatistically reliably suppressed the growth of ascites in animals, incomparison with the control group.

The data on counting the number of live and dead cells in the ascites ofthe experimental animals correlated with the results described above.Thus, at day 13 of the experiment, the absence of ascitic fluid wasnoted in the abdominal cavities of the animals of the therapeutic group,in contrast to the control (4±0.9 ml) (Table 2). The absence of livetumor cells in the abdominal cavities of the mice of the experimentalgroup was also noted (Table 2).

TABLE 2 Total number of cells in the ascites at day 13 after inoculationof EAC cells Group of experimental animals Number of live Number Volumeof cells/mouse of dead ascites (M ± m), (M ± m) cells/mouse ml/mouse EACcontrol 1.05 ± 0.2 × 10⁹ Less than 2% 4 ± 0.9 EAC + ⁶⁴Zn_(e) 0* 0 0*aspartate, 75 μg/mouse *p < 0.05 as compared with the control group

The data show that the composition comprising ⁶⁴Zn_(e) aspartatesuppressed the growth of Ehrlich ascites carcinoma in the experimentalmice by 100%, after 5 injections of the agent. Thus, at day 13 of theexperiment, the absence of ascitic fluid was noted in the abdominalcavities of the animals of the therapeutic group, in contrast to thecontrol (4±0.9 ml) (Table 2). The absence of live tumor cells in theabdominal cavities of the mice of the experimental group was also noted(Table 2).

This indicates that the agent comprising ⁶⁴Zn_(e) aspartate, as well asthe method that provides five-time intraperitoneal administration of thesaid agent, ensures complete suppression of the growth of theexperimental ascetic tumor in the in vivo experiment on EAC model.

Example 8. Toxicological Study of the Agent According to the Inventionfor Acute Toxicity

In the experiment, the acute toxicity effects of the ⁶⁴Zn_(e)composition were assessed and the tolerated dose of the ⁶⁴Zn_(e)compound included in the composition was determined.

To this end, the effects of the agent according to the invention on thebasic physiological functions of healthy animals were studied. 70 maleCs7Bl/6J mice at the age of 7-8 weeks, weighing 18-23 g were used in theexperiment. The animals were taken from R. E. Kavetsky IEPOR NASUVivarium. Before the experiment, all the animals were healthy, withnormal behavioral performance. During the experiment, the animals werekept in plastic cages under natural light illumination, on a standarddiet with free access to food and water.

Clinical observations were carried out by daily examination of allanimals kept in cages to identify mortality or any signs of a deviationin health status. When revealing deviations, a thorough examination wascarried out. All deviations were recorded. The size of the tumors wasmeasured every 2 days (3 times a week) after the apparent onset ofascites/tumor node growth (at day 8 after the inoculation of tumorcells). Dying animals were euthanized, weighed and necropsied. Thedecision to euthanize such animals was taken jointly with the researchsupervisor and veterinarian. The animals were subjected to completenecrosis, which included examination of the external surface of thebody, all passages, cranial, thoracic, abdominal cavities and theircontents.

In the experiment, the animals were administered the solution comprising⁶⁴Zn_(e) aspartate intraperitoneally at doses of 120 μg/mouse, 80μg/mouse, 50 μg/mouse and 25 μg/mouse, and intravenously at doses of 50μg/mouse, 25 gig/mouse and 12.5 μg/mouse. The agent was dissolved indeuterium-depleted water and injected 7 times every other day for 14days in a volume of 0.5 ml/mouse for intraperitoneal injection and in avolume of 0.2 ml for intravenous injection with the agent. In order todetermine the maximum tolerated dose of ⁶⁴Zn_(e) aspartate included inthe composition of the agent according to the invention, the laboratorymice were injected with the ⁶⁴Zn_(e) aspartate solution using a seriesof injections (7 injections) and different routes of administration(i.p. or i.v.). The animals were observed during 24 days after the firstadministration of the agent (14 days for a series of injections of the⁶⁴Zn_(e) compound and 10 days after the last administration of theagent). The overall health of the animals, state of coat and mucousmembranes, their behavior, reflexes, and basic functions of thegastrointestinal tract were evaluated. The data obtained are presentedin Table 3.

TABLE 3 Assessment of the overall health of the experimental animalsduring 24 days of the experiment Group of experimental animals Generalcondition of the experimental animals Control 100% of the animals in thegroup are alive. Locomotor activity is normal. Behavioral reactions arenormal. No signs of lethargy, weakness, convulsions, paralysis,cyanosis, hypersalivation, diarrhea, vomiting, bleeding, urinationdisorders are observed. Condition of the coat and visible mucousmembranes is normal. Respiratory activity is not impaired. +⁶⁴Zn_(e)aspartate 100% of the animals in the group died within 48 hours afterthe administered i.p. first injection. Locomotor activity was impaired.Lethargy, at a dose of 120 weakness, convulsions, paralysis wereobserved. Respiratory μg/mouse activity was impaired. +⁶⁴Zn_(e)aspartate 80% of the animals in the group are alive. Some changes in theadministered i.p. behavior of animals, such as flaccidity, were observedduring 60 at a dose of 80 min. after each injection of the agent. Laterthe general condition μg/mouse of mice returned to normal. Further, thebehavioral responses were normal: no signs of lethargy, weakness,convulsions, paralysis, cyanosis, hypersalivation, vomiting, bleeding orurination disorders were observed. Locomotor activity was normal.Condition of the coat and visible mucousmembranes was normal.Respiratory activity was not impaired. It should be noted that ashort-lasting diarrhea was observed in 40% of the animals in the group18-24 hours after administration of the agent. 20% of the animals in thegroup died after 4 or 5 injections with ⁶⁴Zn_(e) aspartate (on the8^(th) or 10^(th) day after the first injection). +⁶⁴Zn_(e) aspartate100% of the animals in the group are alive. Locomotor activity isadministered i.p. normal. Behavioral reactions are normal. No signs oflethargy, at a dose of 50 weakness, convulsions, paralysis, cyanosis,hypersalivation, μg/mouse diarrhea, vomiting, bleeding, urinationdisorders are observed. Condition of the coat and visible mucousmembranes is normal. Respiratory activity is not impaired. +⁶⁴Zn_(e)aspartate 100% of the animals in the group are alive. Locomotor activityis administered i.p. normal. Behavioral reactions are normal. No signsof lethargy, at a dose of weakness, convulsions, paralysis, cyanosis,hypersalivation, 25 μg/mouse diarrhea, vomiting, bleeding, urinationdisorders are observed. Condition of the coat and visible mucousmembranes is normal. Respiratory activity is not impaired. +⁶⁴Zneaspartate 100% of the animals in the group died within 10-20 minutesafter administered i.v. the first injection. Locomotor activity wasimpaired. Lethargy, at a dose of 50 weakness, convulsions, paralysiswere observed. Respiratory μg/mouse activity was impaired. +⁶⁴Zn_(e)aspartate 100% of the animals in the group are alive. Locomotor activityis administered i.v. normal. Behavioral reactions are normal. No signsof lethargy, at a dose of 25 weakness, convulsions, paralysis, cyanosis,hypersalivation, μg/mouse diarrhea, vomiting, bleeding, urinationdisorders are observed. Conditionof the coat and visible mucousmembranes is normal. Respiratory activity is not impaired. +⁶⁴Zn_(e)aspartate 100% of the animals in the group are alive. Locomotor activityis administered i.v. normal. Behavioral reactions are normal. No signsof lethargy, at a dose of 12.5 weakness, convulsions, paralysis,cyanosis, hypersalivation, μg/mouse diarrhea, vomiting, bleeding,urination disorders are observed. Condition of the coat and visiblemucous membranes is normal. Respiratory activity is not impaired.

As a result, the maximum tolerated doses of the ⁶⁴Zn_(e) aspartatecomposition for differentroutes of administration were selected when100% of the animals remained alive for 24 days (from the moment of thefirst administration of the agent) showing no signs of intoxication. Themaximum tolerated dose of the composition comprising ⁶⁴Zn_(e) aspartateadministered intravenously was 25 μg/mouse while for intraperitonealadministration it was 80 μg/mouse.

During the study of tolerability of the doses of the agent comprising⁶⁴Zn_(e) aspartate used in the experiment, the effect of the injectedagent on the weight of the laboratory mice was also assessed. The dataare presented in Table 4.

TABLE 4 Changes in the weight of experimental animals afteradministration of the agent comprising ⁶⁴Zn_(e) compounds Group/Day ofexperiment 0 2 4 8 Control 21.8 ± 0.3 20.8 ± 0.4 20.3 ± 0.5 21.1 ± 0.5+⁶⁴Zn_(e) aspartate administered 21.4 ± 0.4 20.5 ± 0.5   17.3 ± 0.2**   17.5 ± 0.1*** i.p. at a dose of 80 μg/mouse +⁶⁴Zn_(e) aspartateadministered 21.3 ± 0.5 20.4 ± 0.4 19.2 ± 0.6 19.8 ± 0.7 i.p. at a doseof 50 μg/mouse +⁶⁴Zn_(e) aspartate administered 21.1 ± 0.5 20.1 ± 0.318.3 ± 0.7 18.4 ± 1   i.p. at a dose of 25 μg/mouse +⁶⁴Zn_(e) aspartateadministered 21.8 ± 0.3 21.3 ± 0.5 20.8 ± 0.5 21.4 ± 0.5 i.v. at a doseof 25 μg/mouse +⁶⁴Zn_(e) aspartate administered 21.1 ± 0.4 20.9 ± 0.320.2 ± 0.5 20.6 ± 0.9 i.v. at a dose of 12.5 μg/mouse Group/Day ofexperiment 11 15 19 24 Control 21.3 ± 0.6 20.8 ± 0.6   21 ± 0.5 21.2 ±0.4 +⁶⁴Zn_(e) aspartate administered    17.3 ± 0.1***    16.5 ± 0.1***   17.2 ± 0.2***    17.6 ± 0.1*** i.p. at a dose of 80 μg/mouse+⁶⁴Zn_(e) aspartate administered 19.5 ± 0.8   19 ± 0.6 19.2 ± 0.7   20 ±0.5 i.p. at a dose of 50 μg/mouse +⁶⁴Zn_(e) aspartate administered  18.2± 0.9* 19.2 ± 0.5 19.8 ± 0.4 20.5 ± 0.6 i.p. at a dose of 25 μg/mouse+⁶⁴Zn_(e) aspartate administered 21.4 ± 0.5 21.3 ± 0.6 21.7 ± 0.4 21.6 ±0.5 i.v. at a dose of 25 μg/mouse +⁶⁴Zn_(e) aspartate administered 20.6± 1   20.9 ± 0.9   21 ± 0.7 21.1 ± 0.8 i.v. at a dose of 12.5 μg/mouse*p < 0.05, **p < 0.005, ***p < 0.001 as compared with the control group.

The data in Table 4 show that administration of the agent comprising⁶⁴Zn_(e) isotope as aspartate resulted in insignificant changes inweight of the experimental animals. At the same time, there is astatistically significant (p<0.001) tendency to loss in the full bodyweight of the animals that received i.p. treatment with ⁶⁴Zn_(e)aspartate at a dose of 80 μg/mouse. In this group, the body weight lossby 15-20%, compared to the control figures, was observed in the micealready on the 4^(th) day after the first injection with the agent. Itcan be assumed that administration of ⁶⁴Zn_(e) aspartate at a dose of 80μg/mouse leads to disruption of certain biochemical processes in mice.

Example 9. Studies Supporting the Efficacy of the ⁶⁴Zn_(e) AspartateComposition and Method Performed in In Vivo Experiments Using MouseL1210 Leukemia Model

The efficacy of the ⁶⁴Zn_(e) aspartate composition with respect togrowth and metastatic activity of the experimental tumor (strain ofmouse L1210 leukemia) was assessed in the experiment. Ascitic leukemiccells, strain L1210, obtained from Bank of Cell Lines from Human andAnimal Tissues, R. E. Kavetsky IEPOR NAS of Ukraine, and maintained inDBA2 mice were used in the experiment. To inoculate the strain, tumorcells derived from ascitic fluid were placed in normal saline solution,the cellularity of suspension was determined using a hemocytometer andadjusted to a concentration of 1×10⁶ cells/ml with saline solution. Thetumor was transplanted by injecting 250 μl of the tumor cell suspension(0.25×10⁶ cells/mouse) into abdominal cavities of the animals. 80 maleBDFi mice at the age of 8-10 weeks, weighing 18-22 g were used in theexperiment.

The animals were taken from R. E. Kavetsky IEPOR NASU Vivarium. Beforethe experiment, all the animals were healthy, with normal behavioralperformance. During the experiment, the animals were kept in plasticcages under natural light illumination, on a standard diet with freeaccess to food and water.

In the experiment, 0.25×10⁶ cells of mouse L1210 leukemia in 0.3 ml ofsaline solution were injected in the peritoneal cavities of the BDFimice. The agent according to the invention was dissolved indeuterium-depleted water (deuterium-depleted water Langvey) and given tothe experimental animals from the first day after the inoculation of thetumor cells. The solution composition comprising ⁶⁴Zn_(e) aspartate anddeuterium-depleted water was injected intraperitoneally using amicroinjection syringe at doses of 25 μg/mouse, 50 μg/mouse and 75pig/mouse every other day (7 times during 14 days) in a volume of 0.5ml/mouse. The said agent was also injected intravenously at a dose of 25μg/mouse every other day (7 times during 14 days) in a volume of 0.2ml/mouse. The dynamics of tumor growth in the experimental animals wasobserved for 60 days (by the volume of ascites in the peritonealcavity). At day 8, 13 and 18 after inoculation of tumor cells, the wholeascites fluid was recovered from the animals and the total number oflive/dead cells in each mouse was determined. Some mice were also usedto evaluate their average life span in the experiment.

Clinical observations were carried out as described in Example 7. Theanimals were divided into groups as follows (16 mice per group):

-   -   1. Group No 1: control group, L1210+solvent (deuterium-depleted        water);    -   2. Group No 2: L1210+⁶⁴Zn_(e) aspartate administered i.v. at a        dose of 25 μg/mouse;    -   3. Group No 3: L1210+⁶⁴Zn_(e) aspartate administered i.p. at a        dose of 25 μg/mouse;    -   4. Group No 4: L1210+⁶⁴Zn_(e) aspartate administered i.p. at a        dose of 50 μg/mouse;    -   5. Group No 5: L1210+⁶⁴Zn_(e) aspartate administered i.p. at a        dose of 75 μg/mouse.

⁶⁴Zn_(e) aspartate was diluted in deuterium-depleted water and injected,starting from the first day of the tumor transplantation, 7 times everyother day during 14 days in a volume of 0.5 ml/mouse using an i.p. routeof administration and in a volume of 0.2 ml/mouse using an i.v. route ofadministration. The animals of the control group receivedintraperitoneal injections with 0.5 ml/mouse of deuterium-depletedwater.

The dynamics of tumor growth in the experimental animals was observedfor 60 days following the inoculation of tumor cells (by the volume ofascites in the peritoneal cavity). The dynamics of formation of ascitesin the control and experimental groups was assessed visually using a10-point scale (one point was 0.5 cm of the mean diameter of the mouseabdomen filled with ascites).

In addition, at 8, 13 and 18 days after the mice were inoculated withthe tumor cells, all ascitic fluid was removed from the abdomens in allanimals from each group by washing their peritoneal cavities with salinesolution and live and dead tumor cells were counted using thetraditional vital dye trypan blue (HyClon, USA) and a hemocytometer.

The number of cells was determined using the following formula:

X=((a)/80)×10⁶, where X is the number of cells in 1 ml and a is thenumber of cells counted in 80 small squares of the hemocytometer.

50% of the mice in each group were used to assess their average lengthof survival in the experiment. The percentage change in the lifespan ofthe experimental animals was calculated using the following formula:(c−d)/c×100, where: c is the average lifespan in the experimental groupand d is the average lifespan in the control group. The total time ofobservation of the lifespan of the experimental animals was 60 daysafter the tumor inoculation.

To assess the significance levels of differences in average valuesbetween the groups, the Student's t-test and non-parametric Mann-WhitneyU test were applied. Calculations were performed using Statistica 6.0software package.

As a result of in vivo experiment on the assessment of antitumoractivity of the solution of ⁶⁴Zn_(e) aspartate in deuterium-depletedwater, the following data on the dynamics of ascites growth inexperimental animals were obtained in the mouse L1210 leukemia model:

TABLE 5 Dynamics of L1210 leukemia growth (analysis of changes in thevolumes of ascites in laboratory animals) Group of Day after inoculationof tumor cells experimental 8 10 13 15 18 20 22 26 28 animals Volume ofascites in mice, points L1210 control 2.5 4.7 ± 0.2 8.6 ± 0.3 10 — — — —— L1210 + ⁶⁴ Zn_(e) 3.3 ± 0.3** 4.7 ± 0.4 7.7 ± 0.4 9.7 ± 0.3 8 ± 0.9 10— — — aspartate administered i.v. at a dose of 25 μg/mouse L1210 +⁶⁴Zn_(e) 0.6 ± 0.3* 1.1 ± 0.5* 2.5± 0.6* 4.1 ± 1.1* 4.8 ± 1.4 4.6 ± 1.83.5 ± 2.2 2.5 ± 2.5 0 aspartate administered i.p. at a dose of 25μg/mouse L1210 + ⁶⁴Zn_(e) 0* 0* 0.4 ± 0.2* 1.3 ± 0.7* 1.1 ± 1.1  0 0 0 0aspartate administered i.p. at a dose of 50 μg/mouse L1210 + ⁶⁴Zn_(e) 0*0* 0.4 ± 0.4* 1 ± 1* 1.1 ± 1.1  0 0 0 0 aspartate administered i.p. at adose of 75 μg/mouse *p < 0.001, **p < 0.02 as compared with the controlgroup.

The analysis of data in Table 5 shows that the solution comprising⁶⁴Zn_(e) aspartate and deuterium-depleted water, at dosages selected forthe study, suppresses L1210 leukemia cell growth significantly only whenadministered intraperitoneally. A series of intravenous injections withthe same ⁶⁴Zn_(e) aspartate solution produced no effect on the growth ofmouse leukemia L1210 cells. It should be noted that both in the controland in the experimental groups, where animals received i.v. or i.p.injections with a ⁶⁴Zn_(e) aspartate at a dose of 25 μg/mouse, thegrowth of tumor cells was already recorded at day 8 after the tumorinoculation. However, in the groups where mice were injected with 50g/mouse or 75 g/mouse of the agent, the ascites growth was recorded onlyon the 13^(th) day in the experiment (Table 5).

It was also shown that increasing the dose of the ⁶⁴Zn_(e) aspartate ledto enhancement of its antitumor activity (Table 5). Thus at day 15 afterthe tumor inoculation, while the amount of ascites in all mice in thecontrol group reached the maximum value (10 points), in the group wheremice received i.p. treatment with ⁶⁴Zn_(e) aspartate at a dose of 25g/mouse the amount of ascites was 60% (4.1 points) less, and in thegroups where the experimental animals were injected with 50 μg and 75 μgof the ⁶⁴Zn aspartate solution, the amount of ascites was 87% (1.3points) and 90% (1 point) less respectively.

The data on the number of live and dead cells in the ascites harvestedfrom the peritoneal cavities of the experimental animals were correlatedwith the described above results. Thus at day 8 and day 13 after theinoculation of tumor cells, no ascites in the peritoneal cavities ofanimals in the therapeutic group treated with the agent comprising⁶⁴Zn_(e) aspartate at the doses of 50 μg/mouse or 75 μg/mouse wasobserved, in contrast to the control group (Tables 6, 7). Moreover, theabsence of live tumor cells in the peritoneal cavities of mice of theseexperimental groups was recorded (Tables 6, 7). It should be noted thatthe data presented in Table 6 show that the composition comprising⁶⁴Zn_(e) aspartate suppresses the growth of L1210 leukemia cells after 3injections, as a statistically significant decrease in the number oflive cells, by 100%, in the peritoneal cavities of mice in theexperimental group where animals received the agent comprising ⁶⁴Znaspartate at the doses of 50 μg/mouse or 75 μg/mouse was observed.

TABLE 6 Total number of cells in the ascites at day 8 after inoculationof L1210 cells Group of experimental animals Number of Number of Volumeof live dead ascites cells/mouse cells/mouse (M ± m), (M ± m) (M ± m), %ml/mouse Remarks L1210 control 659.4 ± below 2 1.5 ± 0.3 — 37.8 × 10⁶L1210 + ⁶⁴Zn_(e) 902 ± below 2 1.2 ± 0.3 — aspartate 169 × 10⁶administered i.v. at a dose of 25 μg/mouse L1210 + ⁶⁴Zn_(e) 335.8 ±below 2 0.8 ± 0.8 Of 2 mice in this aspartate 335.8 × 10⁶ experimentalgroup from administered i.p. which ascites was removed, at a dose of 25the presence of fluid with μg/mouse tumor cells was observed in onemouse. Moreover, the number of cells in the ascites of this mouse was atthe level of control indicators (671. 6 × 10⁶). No fluid or tumor cellsin the peritoneal cavity of the other mouse were observed. L1210 +⁶⁴Zn_(e) 0** 0 0* — aspartate administered i.p. at a dose of 50 μg/mouseL1210 + ⁶⁴Zn_(e) 0** 0 0* — aspartate administered i.p. at a dose of 75μg/mouse *p < 0.05, **p < 0.005 as compared with the control group.

TABLE 7 Total number of cells in the ascites at day 13 after inoculationof L1210 cells Group of experimental animals Number of Number of Volumeof live dead ascites cells/mouse cells/mouse (M ± m), (M ± m) (M ± m), %ml/mouse Remarks L1210 control 1403 ± below 3 4 ± 0.4 — 108 × 10⁶L1210 + ⁶⁴Zn_(e) 1322 ± below 3 6 ± 1.5 — aspartate 557 × 10⁶administered i.v. at a dose of 25 μg/mouse L1210 + ⁶⁴Zn_(e) 532.7 ±below 3 1.9 ± 1.5   Of 3 mice in this aspartate 363.8 × 10⁶ experimentalgroup from administered i.p. which ascites was removed, at a dose of 25the presence of fluid with μg/mouse tumor cells was observed in twomice. Moreover, the number of cells in the ascites of the first mousewas at the level of control indicators (1228.2 × 10⁶) and in the secondmouse it was 3.8 times smaller (370 × 10⁶). No fluid or tumor cells inthe peritoneal cavity of the third mouse were observed. L1210 + ⁶⁴Zn_(e)0** 0 0* — aspartate administered i.p. at a dose of 50 μg/mouse L1210+⁶⁴Zn_(e) 0** 0 0* — aspartate administered i.p. at a dose of 75μg/mouse *p < 0.005, **p < 0.001 as compared with the control group.

A realistic assessment of antitumor activity of the agent comprising⁶⁴Zn_(e) aspartate and deuterium-depleted water in the therapeuticgroups was performed by counting the number of tumor-free mice (Table8).

The data presented in Table 8 show that a series of 7 i.p. injectionswith the solution comprising ⁶⁴Zn_(e) aspartate at the dose of 75μg/mouse suppresses the growth of tumor cells in the experimentalanimals by 93.8%, at the dose of 50 μg/mouse by 68.8% and at the dose of25 μg/mouse by 31.2%, compared with the control group.

TABLE 8 Suppression of the growth of L1210 leukemic cells after theirexposure to the action of the solution comprising ⁶⁴Zn_(e) aspartateGroup of experimental animals Number of Number of Tumor- animals in theanimals in the free group with group without animals, tumor, pc tumor,pc % L1210 control 16 0 0 L1210 +⁶⁴Zn_(e) aspartate 16 0 0 administeredi.v. at a dose of 25 μg/mouse L1210 +⁶⁴Zn_(e) aspartate 11 5 31.2administered i.p. at a dose of 25 μg/mouse L1210 +⁶⁴Zn_(e) aspartate 511 68.8 administered i.p. at a dose of 50 μg/mouse L1210 +⁶⁴Zn_(e)aspartate 1 15 93.8 administered i.p. at a dose of 75 μg/mouse

Therapeutic effects of the composition comprising ⁶⁴Zn_(e) aspartate onthe mouse L1210 leukemia model were also assessed taking into accountthe average lifespan of the experimental animals. The following resultswere obtained:

TABLE 9 Increase in the lifespan of experimental animals treated withthe solution comprising ⁶⁴Zn_(e) aspartate Group of experimental animalsAverage lifespan of Increase in the lifespan of animals (M ± m), daysexperimental animals, % L1210 control 16.5 ± 0.8 — L1210 + ⁶⁴Zn_(e)aspartate 16.9 ± 1.3 2.4 administered i.v. at a dose of 25 μg/mouseL1210 + ⁶⁴Zn_(e) aspartate  28.1 ± 4.7* 41.3 administered i.p. at a doseof 25 μg/mouse L1210 + ⁶⁴Zn_(e) aspartate  41.6 ± 8.7* 60.3 administeredi.p. at a dose of 50 μg/mouse L1210 + ⁶⁴Zn_(e) aspartate   55 ± 5** 70administered i.p. at a dose of 75 μg/mouse *p < 0.05, **p < 0.001 ascompared with the control group

The data analysis shows that the agent comprising 6Zn_(e) aspartateadministered intraperitoneally statistically significantly increases theaverage lifespan of the experimental mice (FIG. 1). Thus, compared withthe control group (16.5±0.8 days), the average lifespan of mice thatwere injected with the agent comprising ⁶⁴Zn_(e) aspartate at the doseof 25 gig/mouse increased by 41.3% (28.1±4.7 days), in the group wheremice received the agent at the dose of 50 gig/mouse the average lifespanincreased by 60.3% (41.6±8.7 days) and in the group “L1210+⁶⁴Zn_(e)aspartate administered Lp. at a dose of 75 μg/mouse” this figureincreased by 70% (55±5 days) (Table 9).

Thus the applicant demonstrated the in vivo antitumor activity of the⁶⁴Zn_(e) isotope as aspartate against the models of mouse breast cancer(Ehrlich ascites carcinoma) and mouse leukemia (L1210 strain). A seriesof i.p. injections with the agent according to the inventionsignificantly suppressed the growth of experimental tumors, by 31 to 94%depending on the dosages of the agent comprising ⁶⁴Zn_(e) aspartate, andincreased the average lifespan of experimental animals by 70% (in thecase of administering the agent to mice at a dose of 75 μg/mouse).

An antitumor action of the solution comprising ⁶⁴Zn_(e) aspartate isprobably attributable not to direct but to mediated cytotoxic effect ofthe drug on the tumor cells which is manifested by inhibition of theirproliferation and activation of nonspecific immunity, as the percentageof dead tumor cells in the ascites treated with the agent comprising⁶⁴Zn_(e) aspartate did not exceed 5%. This is also evidenced by thetotal absence of acute toxicity in mice with complete inhibition oftumor growth.

While the invention has been described with reference to exemplaryembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

1. A therapeutic composition comprising an effective amount of one ormore of ³⁹K-enriched potassium, ²⁴Mg-enriched magnesium, ⁶⁴Zn-enrichedzinc, ⁸⁵Rb-enriched rubidium, and ²⁸Si-enriched silicon, present as anelement or in the form of a pharmaceutically acceptable salt, compoundor complex thereof, and at least one excipient.
 2. The composition ofclaim 1, wherein the composition contains ³⁹K-enriched potassium and the³⁹K-enriched potassium is at least 98% ³⁹K.
 3. The composition of claim2, wherein the composition contains between 1 g and 14.5 g of ³⁹K. 4.The composition of claim 1, wherein the composition contains²⁴Mg-enriched magnesium and the ²⁴Mg-enriched magnesium is at least 95%²⁴Mg.
 5. The composition of claim 4, wherein the composition containsbetween 10 mg and 13 g of ²⁴Mg.
 6. The composition of claim 1, whereinthe composition contains ⁶⁴Zn-enriched zinc and the ⁶⁴Zn-enriched zincis at least 90% ⁶⁴Zn.
 7. The composition of claim 6, wherein thecomposition contains between 0.6 mg and 330 mg of 6Zn.
 8. Thecomposition of claim 7, wherein the ⁶⁴Zn-enriched zinc is present as achelate of an amino acid.
 9. The composition of claim 1, wherein thecomposition contains ⁸⁵Rb-enriched rubidium and the ⁸⁵Rb-enrichedrubidium is at least 90% ⁸⁵Rb.
 10. The composition of claim 9, whereinthe composition contains between 0.3 mg and 60 mg of ⁸⁵Rb.
 11. Thecomposition of claim 1, wherein the composition contains ²⁸Si-enrichedsilicon and the ²⁸Si-enriched silicon is at least 95% ²⁸Si.
 12. Thecomposition of claim 11, wherein the composition contains between 3.3 mgand 300 mg of ²⁸Si.
 13. A method of treating a patient having a solidtumor comprising the steps of: administering a composition of claim 1 tothe patient, surgically removing the solid tumor from the patient, andadministering a second composition of claim 1 to the patient.